Phage antibodies to radiation-inducible neoantigens

ABSTRACT

A method for identifying a molecule that binds an irradiated tumor in a subject and molecules identified thereby. The method includes the steps of: (a) exposing a tumor to ionizing radiation; (b) administering to a subject a library of diverse molecules; and (c) isolating from the tumor one or more molecules of the library of diverse molecules, whereby a molecule that binds an irradiated tumor is identified. Also provided are therapeutic and diagnostic methods using targeting ligands that bind an irradiated tumor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/953,780, filed Dec. 10, 2007, which issued as U.S. Pat. No. 8,012,945on Sep. 6, 2011; which is a divisional of Ser. No. 10/689,006, filedOct. 20, 2003, which issued as U.S. Pat. No. 7,306,925 on Dec. 11, 2007;which is a continuation-in-part of co-pending U.S. patent applicationSer. No. 09/914,605, filed Nov. 9, 2001, which issued as U.S. Pat. No.7,049,140 on May 23, 2006; and Ser. No. 10/259,087, filed Sep. 27, 2002,which issued as U.S. Pat. No. 7,402,392 on Jul. 22, 2008, each of whichis herein incorporated by reference in its entirety.

GRANT STATEMENT

This work was supported by grants CA58508, CA68485, CA70937, CA88076,CA89674, CA89888, and CA90949 from the U.S. National Institutes ofHealth. Thus, the U.S. government has certain rights in the presentlydisclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter generally relates to antibodiesthat bind to radiation-inducible targets. More particularly, thepresently disclosed subject matter provides a method for screening aplurality of phage-displayed antibodies for an ability to bind to aradiation-inducible neoantigen present on a cell. Also provided arecompositions comprising scFv antibodies that bind to radiation-inducibletargets.

TABLE OF ABBREVIATIONS

-   -   6×His an epitope tag consisting of six consecutive histidine        residues    -   AR autoradiography    -   CAM cell adhesion molecule    -   CDR complementarity-determining region    -   CEA carcinoembryonic antigen    -   CPM counts per minute    -   CT computerized tomography    -   Dil 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine        perchlorate    -   DOPE dioleoylphosphatidylethanolanime    -   DTPA diethylenetriamine pentaacetate    -   DWI diffusion-weighted imaging    -   EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide    -   EDTA ethylenediamine tetraacetic acid    -   Fc immunoglobulin constant region    -   FMAT fluorometric microvolume assay technology    -   FITC fluorescein isothiocyanate    -   fMRI functional MR imaging    -   GBq gigabecquerels    -   GEE Generalized Estimating Equation    -   GM-CSF granulocyte-macrophage colony stimulating factor    -   GP glycoprotein    -   Gy Grays    -   HCL hydrochloric acid    -   HLA human leukocyte antigen    -   HMPAO hexamethylpropylene amine oxime    -   HNSCC head and neck squamous cell carcinoma    -   HRP horseradish peroxidase    -   HPLC high performance liquid chromatography    -   HSPs high scoring sequence pairs    -   HUVEC human umbilical vein endothelial cells    -   IFN interferon    -   IL interleukin    -   IP imaging plate    -   IPX Internet Packet eXchange    -   LUER low energy high resolution    -   M molar    -   MALDI-MS matrix-assisted laser desorption/ionization coupled        mass spectrometry    -   MBq megabecquerels    -   μCi microcuries    -   mCi millicuries    -   NM nuclear magnetic    -   MRI magnetic resonance imaging    -   MRS proton magnetic resonance spectroscopy    -   MS mass spectrometry    -   MTI magnetization transfer imaging (MTI),    -   Ni-NTA nickel-nitrilotriacetic acid    -   OD optical density    -   PBS phosphate-buffered saline    -   PCR Polymerase Chain Reaction    -   PEG polyethylene glycol    -   PET positron emission spectroscopy    -   PFU plaque forming unit    -   REML restricted/residual maximum likelihood    -   ROI region(s) of interest    -   SAS Statistical Analysis System    -   scFv single chain fragment variable    -   SDS sodium dodecyl sulfate    -   SHNH succinimidyl 6-hydrazinium nicotinate hydrochloride    -   SPDP N-succinimidyl 3-(2-pyridylthio)propionate    -   SPECT single photon emission computed tomography    -   SQUID superconducting quantum interference device    -   T/B tumor-to-background ratio    -   TFA trifluoroacetic acid    -   TNF tumor necrosis factor    -   TUNEL terminal deoxynucleotidyl transferase-mediated nick end        labeling    -   V_(H) heavy chain variable region of an antibody or antibody        fragment    -   V_(L) light chain variable region of an antibody or antibody        fragment

Amino Acid Abbreviations, Codes, and Functionally Equivalent Codons 3-1- Amino Acid Letter Letter Codons Alanine Ala A GCA GCC GCG GCUArginine Arg R AGA AGG CGA CGC CGG CGU Asparagine Asn N AAC AAUAspartic Acid Asp D GAC GAU Cysteine Cys C UGC UGU Glutamic acid Glu EGAA GAG Glutamine Gln Q CAA CAG Glycine Gly G GGA GGC GGG GGU HistidineHis H CAC CAU Isoleucine Ile I AUA AUC AUU Leucine Leu LUUA UUG CUA CUC CUG CUU Lysine Lys K AAA AAG Methionine Met M AUGPhenylalanine Phe F UUC UUU Proline Pro P CCA CCC CCG CCU Serine Ser SACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Tryptophan Trp WUGG Tyrosine Tyr Y UAC UAU Valine Val V GUA GUC GUG GUU

BACKGROUND ART

Tumor-specific drug delivery has the potential to minimize toxicity tonormal tissues and improve the bioavailability of therapeutic agents totumor cells (Hallahan et al., 1995b; Arap et al., 1998). Targetingligands include antibodies and peptides that accumulate in tumors byspecific binding to target molecules present on tumor vasculature,endothelial cells associated with tumor vasculature, and tumor cells.Effective target molecules are generally cell surface receptors or othermolecules present at the exterior of tumor cells such that they areaccessible to targeting ligands (Hallahan et al., 2001a).

Existing site-specific drug delivery systems include ligands thatrecognize a tumor marker such as Her2/neu (v-erb-b2 avian erythroblasticleukemia viral oncogene homologue 2), CEA (carcinoembryonic antigen; Itoet al., 1991), and breast cancer antigens (Manome et al., 1994; Kirpotinet al., 1997; Becerril et al., 1999). See also PCT InternationalPublication No. WO 98/10795. In an effort to identify ligands that arecapable of targeting to multiple tumor types, targeting ligands havebeen developed that bind to target molecules present on tumorvasculature (Baillie et al., 1995; Pasqualini & Ruoslahti, 1996; Arap etal., 1998; Burg et al., 1999; Ellerby et al., 1999).

Despite these advances, current methods for targeted drug delivery arehindered by targeting ligands that also bind normal tissues and/or alack of targeting ligands that bind multiple tumor types. Ideally, atargeting molecule should display specific targeting in the absence ofsubstantial binding to normal tissues, and a capacity for targeting to avariety of tumor types and stages. Thus, there exists a long-felt needin the art for methods to achieve site-specific, tumoral delivery oftherapeutic and/or diagnostic agents.

To meet this need, the presently disclosed subject matter provides amethod for identifying ligands that bind to irradiated tumors. Suchligands are useful for x-ray guided drug delivery, among otherapplications.

SUMMARY

The presently disclosed subject matter provides methods and compositionsthat can be used to target radiation-inducible neoantigens. In oneembodiment, a radiation-inducible neoantigen is selected from the groupconsisting of P-selectin, E-selectin, Endoglin, α_(2b)β₃ integrin, andα_(v)β₃ integrin.

The presently disclosed subject matter also provides a method forscreening a plurality of phage-displayed antibodies for an ability tobind to a radiation-inducible neoantigen present on a cell. In oneembodiment, the method comprises (a) contacting the cell with a firstsolution, the first solution comprising the plurality of phage-displayedantibodies; (b) isolating a second solution, the second solutioncomprising those phage-displayed antibodies that do not bind to thecell; (c) removing any phage-displayed antibodies bound to the cell; (d)treating the cell with radiation, wherein the treating results in aradiation-inducible neoantigen being present on the cell; (e) contactingthe cell with the second solution; and (f) detecting binding of aphage-displayed antibody to the radiation-inducible neoantigen on thecell.

The methods and compositions of the presently disclosed subject matteremploy or comprise antibodies or antibody fragments. In one embodiment,an antibody or antibody fragment is a single chain fragment variable(scFv) antibody. In another embodiment, an antibody or antibody fragmentis a human Fab antibody. Thus, in representative embodiments, themethods and compositions of the presently disclosed subject matteremploy or comprise scFv antibodies or human Fab antibodies.

In one embodiment, the phage-displayed antibody is humanized. In anotherembodiment, the phage-displayed antibody is encoded by a nucleic acidencoding an scFv antibody having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 18, 20, 22, and 24, or by a nucleicacid sequence that is selected from the group consisting of SEQ ID NOs:17, 19, 21, and 23. In another embodiment, the phage-displayed antibodyhas an amino acid sequence that is selected from the group consisting ofSEQ ID NOs: 18, 20, 22, and 24. In still another embodiment, thephage-displayed antibody further comprises an epitope tag. In oneembodiment, the epitope tag is selected from the group consisting of ac-myc tag and a histidine tag.

The present method can be used to screen for phage-displayed antibodiesthat bind to radiation-inducible neoantigens present on any cell. In oneembodiment, the cell is selected from the group consisting of a tumorcell and a vascular endothelial cell. In one embodiment, the vascularendothelial cell is present within tumor microvasculature. In oneembodiment, the radiation-inducible neoantigen is selected from thegroup consisting of P-selectin, E-selectin, Endoglin, α_(2b)β₃ integrin,and α_(v)β₃ integrin.

In one embodiment of the present method, the detecting step isaccomplished using a technique selected from the group consisting ofELISA, BIACORE, Western blotting, immunohistochemistry, fluorometricmicrovolume assay technology, mass spectroscopy, MALDI-MS, andMALDI-TOF.

The presently disclosed subject matter also provides a method oftargeting a therapeutic agent to a target tissue. In one embodiment, themethod comprises (a) providing an immunoconjugate composition comprisinga therapeutic agent and an antibody or antibody fragment, wherein theantibody or antibody fragment is capable of binding to aradiation-inducible neoantigen; (b) irradiating the target tissue toinduce expression of the radiation-inducible neoantigen in the targettissue; and (c) contacting the irradiated target tissue with theimmunoconjugate composition under conditions sufficient for binding ofthe antibody or antibody fragment to the radiation-inducible neoantigen,whereby the therapeutic agent is targeted to the target tissue. In oneembodiment, the target tissue is a tumor or tumor vasculature. Inanother embodiment, the target tissue is present within a subject. Inone embodiment, the subject is a mammal.

The presently disclosed subject matter also provides a method forsuppressing the growth of a tumor in a subject. In one embodiment, themethod comprises (a) exposing the tumor to radiation, whereby aradiation-inducible neoantigen is expressed; and (b) administering tothe subject bearing the tumor an effective amount of an immunoconjugatecomposition, the immunoconjugate composition comprising a therapeuticagent and an antibody or antibody fragment that binds to aradiation-inducible neoantigen, whereby growth of the tumor issuppressed.

The present method can be used to suppress the growth of any tumor inany subject. In one embodiment, the tumor is selected from the groupconsisting of benign intracranial meningiomas, arteriovenousmalformation, angioma, macular degeneration, melanoma, adenocarcinoma,malignant glioma, prostatic carcinoma, kidney carcinoma, bladdercarcinoma, pancreatic carcinoma, thyroid carcinoma, lung carcinoma,colon carcinoma, rectal carcinoma, brain carcinoma, liver carcinoma,breast carcinoma, ovary carcinoma, solid tumors, solid tumor metastases,angiofibromas, retrolental fibroplasia, hemangiomas, Karposi's sarcoma,head and neck carcinomas, and combinations thereof. In one embodiment,the subject is a mammal.

The presently disclosed subject matter also provides a single chainfragment variable (scFv) antibody isolated by the disclosed methods.

The presently disclosed subject matter also provides immunoconjugatecompositions for use in the disclosed methods. In one embodiment, theimmunoconjugate composition comprises an antibody or antibody fragmentthat binds to a radiation-inducible neoantigen. In another embodiment,the immunoconjugate composition comprises a liposome or a nanoparticle.In one embodiment, the nanoparticle further comprises a fluorescentlabel.

In one embodiment, the immunoconjugate composition is polyvalent. Inthis embodiment, the immunoconjugate composition comprises a pluralityof single chain fragment variable (scFv) antibodies, the pluralitycomprising at least two single chain fragment variable (scFv) antibodiesthat bind to different epitopes. In this embodiment, the plurality ofsingle chain fragment variable (scFv) antibodies comprises at least onescFv antibody that binds to an antigen present on a tumor cell and atleast one scFv antibody that binds to an antigen present on a vascularendothelial cell.

In another embodiment of the presently disclosed subject matter, theimmunoconjugate composition further comprises a therapeutic agent. Inone embodiment, the therapeutic agent is selected from the groupconsisting of a virus, a radionuclide, a cytotoxin, a therapeutic gene,and a chemotherapeutic agent.

In another embodiment of the present immunoconjugate composition, thesingle chain fragment variable (scFv) antibody is humanized. In anotherembodiment, the single chain fragment variable (scFv) antibody isencoded by a nucleic acid encoding an scFv antibody having an amino acidsequence selected from the group consisting of SEQ ID NOs: 18, 20, 22,and 24, or by a nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 17, 19, 21, and 23. Instill another embodiment, the single chain fragment variable (scFv)antibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 18, 20, 22, and 24. In one embodiment, theimmunoconjugate composition is provided in a pharmaceutically acceptablecarrier.

In another embodiment, the immunoconjugate composition further comprisesa detectable label. In one embodiment, the detectable label isdetectable in vivo. In this embodiment, the detectable label comprises alabel that can be detected using magnetic resonance imaging,scintigraphic imaging, ultrasound, or fluorescence. In one embodiment,the label that can be detected using scintigraphic imaging comprises aradionuclide label. In this embodiment, the radionuclide label comprises¹³¹I or ^(99m)Tc.

The presently disclosed subject matter also provides a polyvalentimmunoconjugate composition, the polyvalent immunoconjugate compositioncomprising a plurality of single chain fragment variable (scFv)antibodies, wherein the plurality of single chain fragment variable(scFv) antibodies bind to a plurality of different epitopes, and whereinat least one of the epitopes is present on a radiation-inducibleneoantigen. In one embodiment, at least one of the plurality ofdifferent epitopes is present on a vascular endothelial cell.

The presently disclosed subject matter also provides a method forprioritizing the binding of a plurality of antibodies or antibodyfragments to a target tissue in a subject. In one embodiment, the methodcomprises (a) providing a plurality of antibodies or antibody fragmentsthat bind to the target, wherein the plurality of antibodies or antibodyfragments comprise at least two different antibodies or antibodyfragments that bind a radiation-inducible neoantigen within the targettissue, and wherein the at least two different antibodies or antibodyfragments are distinguishable from each other; (b) irradiating thetarget tissue, whereby the radiation-inducible neoantigens are expressedwithin the target tissue; (c) administering the plurality of antibodiesor antibody fragments to the subject under conditions sufficient toallow the plurality of antibodies or antibody fragments to bind to theradiation-inducible neoantigens in the target tissue; (d) isolating aportion of the target tissue from the subject, wherein the portioncomprises the radiation-inducible neoantigens to which the plurality ofantibodies or antibody fragments bind; (e) identifying the at least twodifferent antibodies or antibody fragments in the portion of the targettissue; (f) comparing a relative selectivity and an affinity for theradiation-inducible neoantigens of the at least two different antibodiesor antibody fragments identified in step (e) in the irradiated targettissue; and (g) assigning a priority to the at least two differentantibodies or antibody fragments based on the comparing of step (f). Inone embodiment, the subject is a mammal. In one embodiment, the targettissue is a tumor or tumor vasculature. In one embodiment, theantibodies or antibody fragments are single chain fragment variable(scFv) antibodies. In one embodiment, the single chain fragment variable(scFv) antibodies are humanized. In one embodiment of the presentmethod, the at least two different antibodies or antibody fragments thatbind to at least two different radiation-inducible neoantigens aredistinguishable from each other based upon differences in molecularweight.

In another embodiment of the present method, the at least two differentantibodies or antibody fragments that bind to at least two differentradiation-inducible neoantigens within the target tissue each furthercomprises a different detectable label, such that the antibodies orantibody fragments that bind to different radiation-inducibleneoantigens can be distinguished from each other.

In one embodiment, the different detectable labels are fluorescentlabels, and each fluorescent label has a different excitation oremission spectrum, such that the different antibodies can bedistinguished from each other.

In another embodiment of the present method, the administering is byintravenous injection or intratumoral injection. In another embodiment,the portion of the target tissue from the subject is a tumor biopsy. Instill another embodiment, the detecting is by mass spectroscopy.

The presently disclosed subject matter also provides methods ofdetecting a tumor in a subject. In one embodiment, the method comprises(a) exposing a suspected tumor to ionizing radiation; (b) administeringto the subject an immunoconjugate composition, wherein theimmunoconjugate composition comprises an antibody or antibody fragmentthat binds to a radiation-inducible neoantigen and a detectable label;and (c) detecting the detectable label, whereby a tumor is diagnosed. Inanother embodiment, the method comprises (a) exposing a suspected tumorto ionizing radiation; (b) removing a portion of the suspected tumor;(c) contacting an immunoconjugate composition with the suspected tumorin vitro, wherein the immunoconjugate composition comprises an antibodyor antibody fragment that binds to a radiation-inducible neoantigen anda detectable label; and (d) detecting the detectable label, whereby atumor is diagnosed.

Accordingly, it is an object of the presently disclosed subject matterto provide a method for screening a plurality of antibodies or antibodyfragments for an ability to bind to a radiation-inducible neoantigenpresent on a cell. This and other objects are achieved in whole or inpart by the presently disclosed subject matter.

An object of the presently disclosed subject matter having been statedabove, other objects and advantages of the presently disclosed subjectmatter will become apparent to those of ordinary skill in the art aftera study of the following description and non-limiting Examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a polyvalent immunoconjugate. In this Figure, thepolyvalent immunoconjugate consists of a nanoparticle to which twoantibodies (Antibody 1 and Antibody 2) and one therapeutic agent (inthis case, a gamma emitter) are complexed.

FIG. 2 depicts the output from the FMAT™ 8100 device (PE Biosystems,Foster City, Calif., United States of America) used to prioritizeseveral E-tagged anti-P-selectin single chain fragment variable (scFv)antibodies. In this Figure, Lane F is P-selectin alone. Lane G isP-selectin and secondary (anti-E-tag) antibody. Lanes H-P show thebinding of serial dilutions of 9 different anti-P-selectin scFvantibodies.

FIG. 3 depicts a general strategy for MALDI-TOF Mass Spectrometry ofantibody binding to a target tissue. As depicts in this Figure, acrystalline organic matrix is applied to frozen section of a targettissue that has been treated with a composition of the presentlydisclosed subject matter (either an antibody or fragment thereof, or animmunoconjugate). The matrix-containing sections are then analyzed byMALDI-TOF, which assigns mass-to-charge (m/z) ratios to the desorbedions. The mass image profiles can then be used to generate informationabout the binding of the antibodies or immunoconjugates in the targettissue.

FIG. 4A depicts an illustration and mass spectrophotometric analysis ofaffinity purified scFvs specific for an antigen. In FIG. 4A, arepresentative E-tagged scFv specific for an antigen (in one embodiment,a radiation-inducible neoantigen) is depicted. The presence of the E-tagallows the E-tagged scFv antibody to be affinity purified by using ananti-E-tag monoclonal antibody conjugated to a solid support (forexample, a bead). The scFv depicted in this Figure also has its cognateantigen bound to it, which would allow the affinity purification of boththe scFv and the antigen.

FIGS. 4B and 4C depicts mass spectrophotometric analyses of lysates ofan antibody (G12) immunoprecipitated with the beads depicted in FIG. 4A.The precipitated antibodies were placed directly onto MALDI plates andseparated by mass spectroscopy. Shown are the spectra from negativecontrol cells with no antibody (B) and an antibody precipitated bymonoclonal Anti-E antibody beads (C).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-13 are the amino acid sequences of representative peptideligands that bind to radiation-inducible neoantigens.

SEQ ID NOs: 14 and 15 are the nucleotide sequences of PCR primers thatare used to amplify the nucleic acids encoding recombinant phage.

SEQ ID NO: 16 is the amino acid sequence of a peptide that binds to theradiation-inducible neoantigen, α_(2b)β₃ integrin.

SEQ ID NOs: 17-24 are the nucleic acid and amino acid sequences ofrepresentative scFv antibodies that bind to radiation-inducibleneoantigens. Among SEQ ID NOs: 17-24, the odd numbered sequences arenucleic acid sequences, and the even numbered sequences are the aminoacid sequences that are encoded by the immediately previous SEQ ID NO.Thus, SEQ ID NO: 18 is the amino acid sequence of the scFv antibodyencoded by SEQ ID NO: 17; SEQ ID NO: 20 is the amino acid sequence ofthe scFv antibody encoded by SEQ ID NO: 19; etc.

SEQ ID NO: 25 is the amino acid sequence of the E tag epitope tag.

SEQ ID NOs: 26-34 are the amino acid sequences of peptides that are usedin binding assays targeting the radiation-inducible neoantigen α_(2b)β₃integrin. SEQ ID NOs: 26-30 are the amino acid sequences of certainderivatives of SEQ ID NO: 16. SEQ ID NOs: 31-34 are the amino acidsequences of control peptides. These SEQ ID NOs. are summarized below:

SEQ  ID NO: AMINO ACID SEQUENCE 26 HHLGGAKQAGDV-SGSGS 27SGSGS-HHLGGAKQAGDVC 28 HHLGGAKQAGDV-SGSGS-YYYYY 29 HHLGGAKQAGDV-SGSGSC30 HHLGGAKQAGDV-SGSGSK 31 SGSGS 32 SGSGS-YYYYY 33SGSGSGSSGSGSSGSGS-YYYYY 34 SGSGSSGSGSGS-SGSGS

DETAILED DESCRIPTION I. Definitions

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art; references to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques which would be apparent to one of skill in theart. While the following terms are believed to be well understood by oneof ordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” mean “one or more” when used in this application, including theclaims.

As used herein, unless specifically indicated otherwise, the word “or”is used in the “inclusive” sense of “and/or” and not the “exclusive”sense of “either/or”.

The term “ligand” as used herein refers to a molecule or other chemicalentity having a capacity for binding to a target. A ligand can comprisea peptide, an oligomer, a nucleic acid (e.g., an aptamer), a smallmolecule (e.g., a chemical compound), an antibody or fragment thereof, anucleic acid-protein fusion, and/or any other affinity agent.

The term “small molecule” as used herein refers to a compound, forexample an organic compound, with a molecular weight in one example ofless than about 1,000 daltons, in another example less than about 750daltons, in another example less than about 600 daltons, and in yetanother example less than about 500 daltons. A small molecule also has acomputed log octanol-water partition coefficient that in one example isin the range of about −4 to about +14, and in another example is in therange of about −2 to about +7.5.

The term “control tissue” as used herein refers to a site suspected tosubstantially lack binding and/or accumulation of an administeredligand. For example, in accordance with the methods of the presentlydisclosed subject matter, control tissues include, but are not limitedto a non-irradiated tumor, a non-cancerous tissue, and vascularendothelium that is either non-irradiated or not associated with atumor.

The term “target tissue” as used herein refers to an intended site foraccumulation of a ligand following administration to a subject. Forexample, the methods of the presently disclosed subject matter employ atarget tissue comprising an irradiated tumor or the vasculatureassociated with an irradiated tumor.

The terms “target” or “target molecule” as used herein each refer to anysubstance that is specifically bound by a ligand. Thus, the term “targetmolecule” encompasses macromolecules including, but not limited toproteins, nucleic acids, carbohydrates, lipids, and complexes orcombinations thereof.

As used herein, the terms “radiation-inducible target”,“radiation-inducible tumor target”, and “radiation-inducible neoantigen”are used interchangeably and refer to a target molecule associated witha target tissue (for example, a tumor) for which the expression,localization, or ligand-binding capacity is induced by radiation. Such atarget molecule can comprise a molecule at the surface of a tumor cell,within a tumor cell, or in the extracellular matrix surrounding a tumorcell. Alternatively, a target molecule can comprise a molecule presentat the surface of or within a vascular endothelial cell, or at thesurface of or within a blood component such as a platelet or aleukocyte. Radiation-inducible neoantigens include, but are not limitedto P-selectin, E-selectin, Endoglin, α_(2b)β₃ integrin, and α_(2b)β₃integrin.

The term “induce”, as used herein to refer to changes resulting fromradiation exposure, encompasses activation of gene transcription orregulated release of proteins from cellular storage reservoirs to cellsurfaces. Alternatively, induction can refer to a process ofconformational change, also called activation, such as that displayed bythe glycoprotein IIb/IIIa integrin receptor upon radiation exposure(Staba et al., 2000; Hallahan et al., 2001a). See also U.S. Pat. No.6,159,443.

The terms “targeting” or “homing”, as used herein to describe the invivo activity of a ligand following administration to a subject, referto the preferential movement and/or accumulation of a ligand in a targettissue as compared to a control tissue.

The terms “selective targeting” of “selective homing” as used hereineach refer to a preferential localization of a ligand that results in anamount of ligand in a target tissue that is in one example about 2-foldgreater than an amount of ligand in a control tissue, in another exampleabout 5-fold or greater, and in yet another example about 10-fold orgreater. The terms “selective targeting” and “selective homing” alsorefer to binding or accumulation of a ligand in a target tissueconcomitant with an absence of targeting to a control tissue, in oneembodiment the absence of targeting to all control tissues.

The term “absence of targeting” is used herein to describe substantiallyno binding or accumulation of a ligand in all control tissues where anamount of ligand would be expected to be detectable, if present.

The terms “targeting ligand”, “targeting molecule”, “homing ligand”, and“homing molecule” as used herein each refer to a ligand that displaystargeting activity. In one embodiment, a targeting ligand displaysselective targeting. In another embodiment, a targeting ligand comprisesa phage-displayed antibody (for example, a phage-displayed scFv antibodyor a phage-displayed antibody fragment such as an Fab antibody).

The term “binding” refers to an affinity between two molecules, forexample, a ligand and a target molecule. As used herein, the term“binding” refers to a specific binding of one molecule for another in amixture of molecules. The binding of a ligand to a target molecule canbe considered specific if the binding affinity is about 1×10⁴ M⁻¹ toabout 1×10⁶ M⁻¹ or greater. Thus, the binding of an antibody to anantigen can be thought of as having at least two components: anaffinity, which refers to the strength at which the antibody binds anantigen, and a specificity, which refers to the level ofcross-reactivity an antibody displays between closely related antigens.

The phrase “specifically (or selectively) binds”, when referring to thebinding capacity of a ligand, refers to a binding reaction which isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biological materials. The phrase“specifically binds” also refers to selective targeting, as definedherein above.

The phases “substantially lack binding” or “substantially no binding”,as used herein to describe binding of a ligand in a control tissue,refers to a level of binding that encompasses non-specific or backgroundbinding, but does not include specific binding.

The term “tumor” as used herein refers to both primary and metastasizedsolid tumors and carcinomas of any tissue in a subject, including butnot limited to breast; colon; rectum; lung; oropharynx; hypopharynx;esophagus; stomach; pancreas; liver; gallbladder; bile ducts; smallintestine; urinary tract including kidney, bladder and urothelium;female genital tract including cervix, uterus, ovaries (e.g.,choriocarcinoma and gestational trophoblastic disease); male genitaltract including prostate, seminal vesicles, testes and germ cell tumors;endocrine glands including thyroid, adrenal, and pituitary; skin (e.g.,hemangiomas and melanomas), bone or soft tissues; blood vessels (e.g.,Kaposi's sarcoma); brain, nerves, eyes, head and neck (e.g. head andneck squamous cell carcinomas; HNSCC) and meninges (e.g., astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas and meningiomas). The term “tumor” also encompasses solidtumors arising from hematopoietic malignancies such as leukemias,including chloromas, plasmacytomas, plaques and tumors of mycosisfungoides and cutaneous T-cell lymphoma/leukemia, and lymphomasincluding both Hodgkin's and non-Hodgkin's lymphomas.

The term “subject” as used herein refers to any invertebrate orvertebrate species. The methods of the presently disclosed subjectmatter are particularly useful in the treatment and diagnosis ofwarm-blooded vertebrates. Thus, the presently disclosed subject matterconcerns mammals and birds. More particularly contemplated is thetreatment and/or diagnosis of mammals such as humans, as well as thosemammals of importance due to being endangered (such as Siberian tigers),of economic importance (animals raised on farms for consumption byhumans) and/or social importance (animals kept as pets or in zoos) tohumans, for instance, carnivores other than humans (such as cats anddogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle,oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Alsocontemplated is the treatment of birds, including the treatment of thosekinds of birds that are endangered, kept in zoos, as well as fowl, andmore particularly domesticated fowl, e.g., poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomic importance to humans. Thus, contemplated is the treatment oflivestock, including, but not limited to, domesticated swine (pigs andhogs), ruminants, horses, poultry, and the like.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dose (e.g. radiation dose), etc. ismeant to encompass variations of in one example ±20% or ±10%, in anotherexample ±5%, in another example ±1%, and in yet another example ±0.1%from the specified amount, as such variations are appropriate to performthe disclosed methods.

II. X-Ray Guided Drug Delivery

Ionizing radiation induces proteins in tumor vascular endotheliumthrough transcriptional induction and/or posttranslational modificationof cell adhesion molecules such as integrins (Hallahan et al., 1995a;Hallahan et al., 1996; Hallahan et al., 1998; Hallahan & Virudachalam,1999). For example, radiation induces activation of the integrinα_(2b)β₃, also called the fibrinogen receptor, on platelets. Radiationalso induces the translocation of P-selectin from storage reservoirs invascular endothelium to the vascular lumen. The induced molecules canserve as binding sites for targeting ligands, for example, scFvantibodies.

Several radiation-inducible molecules within tumor blood vessels havebeen identified and characterized including, but not limited toP-selectin, E-selectin, Endoglin, α_(2b)β₃ integrin, and α_(2b)β₃integrin. ¹³¹I-labeled fibrinogen binds specifically to tumors followingexposure to ionizing radiation (U.S. Pat. No. 6,159,443). Peptideswithin fibrinogen that bind to the radiation-inducible α_(2b)β₃ integrininclude HHLGGAKQAGDV (SEQ ID NO: 16) and the RGD peptide (Hallahan etal., 2001a).

The presently disclosed subject matter includes a study of the targetingactivity of ligands that bind to radiation-inducible neoantigens (forexample, ligands that bind to P-selectin or the α_(2b)β₃ integrin) intumor-bearing subjects. Example 1 describes x-ray-guided drug deliveryin animal models using ligand-conjugated liposomes and microspheres.Clinical trials using a radiolabeled α_(2b)β₃ ligand support thefeasibility of x-ray-guided drug delivery in humans, as described inExample 2. See also Hallahan et al., 2001a.

III. Identification of Ligands that Bind Irradiated Tumors

Approaches for optimizing peptide binding affinity and specificity haveincluded modification of peptide conformation and addition of flankingamino acids to extend the minimal binding motif. For example, aminoacids C-terminal to the RGD sequence are differentially conserved inRGD-containing ligands, and this variation correlates with differencesin binding specificity (Cheng et al., 1994; Koivunen et al., 1994).Similarly, cyclization of a prototype RGD peptide to restrict itsconformational flexibility improved interaction of the peptide with thevitronectin receptor, yet nearly abolished interaction with thefibronectin receptor (Pierschbacher & Ruoslahti, 1987).

Despite conservation of binding motifs among ligands that bindirradiated tumors and recognition of factors that can influence ligandbinding, design of peptide sequences for improved targeting activity isyet unpredictable. Approaches for identifying such peptides havetherefore relied on high volume screening methods to select effectivemotifs from peptide libraries (Koivunen et al., 1993; Healy et al.,1995). However, the utility of in vitro-selected peptides isunpredictable in so far as peptide-binding properties are notconsistently recapitulated in vivo. To obviate these challenges, thepresently disclosed subject matter provides a method for in vivoselection of targeting ligands, described further herein below.

Using the in vivo selection method disclosed herein, novel targetingligands were identified that can be used for x-ray-guided drug delivery.Representative peptide ligands are set forth as SEQ ID NOs: 1-13.Representative scFv antibody ligands are set forth as SEQ ID NOs: 18,20, 22, and 24 (encoded by SEQ ID NOs: 17, 19, 21, and 23). The novelligands display improved specificity of binding to irradiated tumors andare effective for targeting using low dose irradiation. The disclosedtargeting ligands also offer benefits including moderate cost ofpreparation and ease of handling.

A. Libraries

As used herein, the term “library” means a collection of molecules. Alibrary can contain a few or a large number of different molecules,varying from at least two molecules to several billion molecules ormore. A molecule can comprise a naturally occurring molecule, or asynthetic molecule that is not found in nature. Optionally, a pluralityof different libraries can be employed simultaneously for in vivoscreening.

Representative libraries include but are not limited to a peptidelibrary (U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409),an oligomer library (U.S. Pat. Nos. 5,650,489 and 5,858,670), an aptamerlibrary (U.S. Pat. Nos. 6,180,348 and 5,756,291), a small moleculelibrary (U.S. Pat. Nos. 6,168,912 and 5,738,996), a library ofantibodies or antibody fragments (for example, an scFv library or an Fabantibody library; U.S. Pat. Nos. 6,174,708, 6,057,098, 5,922,254,5,840,479, 5,780,225, 5,702,892, and 5,667988), a library of nucleicacid-protein fusions (U.S. Pat. No. 6,214,553), and a library of anyother affinity agent that can potentially bind to an irradiated tumor(e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538). In oneembodiment, a library is a phage-displayed antibody library. In anotherembodiment, a library is a phage-displayed scFv library. In anotherembodiment, a library is a phage-displayed Fab library. In still anotherembodiment, a library is a soluble scFv antibody library.

The molecules of a library can be produced in vitro, or they can besynthesized in vivo, for example by expression of a molecule in vivo.Also, the molecules of a library can be displayed on any relevantsupport, for example, on bacterial pili (Lu et al., 1995) or on phage(Smith, 1985).

A library can comprise a random collection of molecules. Alternatively,a library can comprise a collection of molecules having a bias for aparticular sequence, structure, conformation, or in the case of anantibody library, can be biased in favor of antibodies that bind to aparticular antigen or antigens (for example, a radiation-inducibleneoantigen). See e.g., U.S. Pat. Nos. 5,264,563 and 5,824,483. Methodsfor preparing libraries containing diverse populations of various typesof molecules are known in the art, for example as described in U.S.patents cited herein above. Numerous libraries are also commerciallyavailable.

1. Peptide Libraries

In one embodiment, a peptide library comprises peptides comprising threeor more amino acids, in another example at least five, six, seven, oreight amino acids, in another example up to 50 amino acids or 100 aminoacids, and in yet another example up to about 200 to 300 amino acids.

The peptides can be linear, branched, or cyclic, and can includenon-peptidyl moieties. The peptides can comprise naturally occurringamino acids, synthetic amino acids, genetically encoded amino acids,non-genetically encoded amino acids, and combinations thereof.

A biased peptide library can also be used, a biased library comprisingpeptides wherein one or more (but not all) residues of the peptides areconstant. For example, an internal residue can be constant, so that thepeptide sequence is represented as:(XAA₁)_(m)−(AA)₁−(XAA₂)_(n)

where XAA₁ and XAA₂ are any amino acid, or any amino acid exceptcysteine, wherein XAA₁ and XAA₂ are the same or different amino acids, mand n indicate a number XAA residues, wherein m and n are independentlychosen from the range of 2 residues to 20 residues in one embodiment,and from the range of 4 residues to 9 residues in another embodiment,and AA is the same amino acid for all peptides in the library. In oneexample, AA is located at or near the center of the peptide. Morespecifically, in one example m and n are not different by more than 2residues; in another example m and n are equal.

In one embodiment, a library is employed in which AA is tryptophan,proline, or tyrosine. In another embodiment, AA is phenylalanine,histidine, arginine, aspartate, leucine, or isoleucine. In anotherembodiment, AA is asparagine, serine, alanine, or methionine. In stillanother embodiment, AA is cysteine or glycine.

A biased library used for in vivo screening also includes a librarycomprising molecules previously selected by in vitro screening methods.See Example 8.

In one embodiment of the presently disclosed subject matter, the methodfor in vivo screening is performed using a phage peptide library. Phagedisplay is a method to discover peptide ligands while minimizing andoptimizing the structure and function of proteins. Phage are used as ascaffold to display recombinant libraries of peptides and provide avehicle to recover and amplify the peptides that bind to putativereceptor molecules in vivo. In vivo phage selection simultaneouslyprovides positive and subtractive screens based on the spatialseparation of normal tissues and tumors. Phage that specifically bindthe vasculature of normal tissues are removed while specific phage thatbind target molecules present in irradiated tumors are enriched throughserial rounds of bioscreening.

The T7 phage has an icosahedral capsid made of 415 proteins encoded bygene 10 during its lytic phase. The T7 phage display system has thecapacity to display peptides up to 15 amino acids in size at a high copynumber (415 per phage). Unlike filamentous phage display systems,peptides displayed on the surface of T7 phage are not capable of peptidesecretion. T7 phage also replicate more rapidly and are extremely robustwhen compared to other phage. The stability allows for bioscreeningselection procedures that require persistent phage infectivity.Accordingly, the use of T7-based phage display is an aspect of oneembodiment of the presently disclosed subject matter. Example 4describes a representative method for preparation of a T7 phage peptidelibrary that can be used to perform the in vivo screening methodsdisclosed herein.

A phage peptide library to be used in accordance with the screeningmethods of the presently disclosed subject matter can also beconstructed in a filamentous phage, for example M13 or an M13-derivedphage. In one embodiment, the encoded antibodies are displayed at theexterior surface of the phage, for example by fusion to the product ofM13 gene III. Methods for preparing M13 libraries can be found inSambrook & Russell, 2001, among other places.

2. Phage Antibody Libraries

In one embodiment, a ligand that binds to a radiation-inducibleneoantigen is an antibody, or a fragment thereof. To identify antibodiesand fragments thereof that bind to radiation-inducible neoantigens,libraries can be screened using the methods disclosed herein. Librariesthat can be screened using the disclosed methods include, but are notlimited to libraries of phage-displayed antibodies and antibodyfragments, and libraries of soluble antibodies and antibody fragments.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. In a two-chain Fv species, this regionconsists of a dimer of one heavy and one light chain variable domain intight, non-covalent association. In a single-chain Fv species (scFv),one heavy and one light chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the threecomplementarity-determining regions (CDRs) of each variable domaininteract to define an antigen-binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site. For a review of scFv, see Pluckthun, 1994.

The term “antibodies and fragments thereof”, and grammatical variationsthereof, refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules; i.e., molecules that contain anantigen-binding site that specifically bind an antigen. As such, theterm refers to immunoglobulin proteins, or functional portions thereof,including polyclonal antibodies, monoclonal antibodies, chimericantibodies, hybrid antibodies, single chain antibodies (e.g., a singlechain antibody represented in a phage library), mutagenized antibodies,humanized antibodies, and antibody fragments that comprise an antigenbinding site (e.g., Fab and Fv antibody fragments). Thus, “antibodiesand fragments thereof” include, but are not limited to monoclonal,chimeric, recombinant, synthetic, semi-synthetic, or chemically modifiedintact antibodies having for example Fv, Fab, scFv, or F(ab)₂ fragments.

The immunoglobulin molecules of the presently disclosed subject mattercan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass of immunoglobulinmolecule. In one embodiment, the antibodies are human antigen-bindingantibody fragments of the presently disclosed subject matter andinclude, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd, single-chainFvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) andfragments comprising either a V_(L) or V_(H) domain. Antigen-bindingantibody fragments, including single-chain antibodies, can comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the presently disclosed subject matter are antigen-bindingfragments comprising any combination of variable region(s) with a hingeregion, CH1, CH2, and CH3 domains.

The antibodies and fragments thereof of the presently disclosed subjectmatter can be from any animal origin including birds and mammals. Forexample, the antibodies can be human, murine (e.g., mouse and rat),donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As usedherein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598.

In another embodiment of the presently disclosed subject matter, anantibody library (for example, a library of scFv antibodies) can be usedto perform the disclosed screening methods. In this embodiment, a ligandthat binds to an irradiated tumor is an antibody or a fragment thereofthat binds a radiation-inducible neoantigen. Antibodies that bindradiation-inducible neoantigens can be identified by screening a phageantibody library, as described in Examples 8 and 16. Such a library canbe constructed, for example, in M13 or an M13-derived phage. See e.g.,U.S. Pat. Nos. 6,593,081; 6,225,447; 5,580,717; and 5,702,892, allincorporated by reference herein.

Phage-displayed recombinant antibodies are genetically cloned andexpressed on the tip of the M13 bacteriophage (McCafferty et al., 1990).M13 phage infects E. coli that carry an F′ episome (plasmid) andconstantly produce and secrete intact M13 virus particles without lysingthe host cell. The components of the M13 phage include phage DNA, coatproteins, gene III attachment proteins, and other proteins that arefused to the phage proteins. There are 3-5 copies of the gene IIIattachment proteins located on the exterior of the phage that areresponsible for phage attachment to receptors on E. coli cells.

In one embodiment, M13 phage-displayed recombinant antibodies can becreated by linking DNA from antibody-producing B lymphocytes to thephage gene III DNA using the pCANTAB vector (see Example 8). Theproteins encoded by the antibody in gene III DNA are fused to oneanother to produce an antibody-gene III fusion protein. A bacteriophagecarrying the gene fusion will simultaneously contain the antibody DNAand express an antibody molecule on the gene III protein.

A representative, non-limiting approach to obtain and characterizeantigen-specific recombinant antibodies or antibody fragments (forexample, scFv antibodies or human Fab antibodies) is as follows. Phageantibody selections can be performed using antigens immobilized on solidsupports or biotinylated antigens and streptavidin magnetic beads. Analiquot of a phage antibody library can be applied to the antigen.Nonspecific phage antibodies are thereafter washed off of the antigen,and phage that encode bound antibodies can be eluted and used to infectE. coli. Infected E. coli can be plated and rescued with helper phage toproduce an antigen-enriched phage antibody library. The antigen-enrichedlibrary (i.e., a library pre-selected for binding to a particularantigen of interest) can be used in a second round of selection forbinding to the antigen. Subsequent rounds of selection on antigen andhelper phage rescue can be used until the desired antigen-specificantibodies are obtained. Colonies stemming for phage antibody selectionscan be picked from agar plates manually or by using a colony picker (forexample, the QPix2 Colony Picker from Genetix USA Inc., Boston, Mass.,United States of America). Picked colonies can then transferred toappropriate vessels, for example microwell plates, and can be used toproduce soluble recombinant antibodies. See e.g. Example 8.

Phage-displayed recombinant antibodies have several advantages overpolyclonal antibodies or hybridoma-derived monoclonal antibodies.Phage-displayed antibodies can be generated within 8 days. Recombinantantibody clones can be easily selected by panning a population ofphage-displayed antibodies against immobilized antigen (McCafferty etal., 1990). The antibody protein and antibody DNA are simultaneouslycontained in one phage particle (Better et al., 1988). Liters ofphage-displayed recombinant antibodies can be produced inexpensivelyfrom bacterial culture supernatant and the phage antibodies can be useddirectly in immunoassays without purification. Phage display technologymakes possible the direct isolation of monovalent scFv antibodies. Thesmall size of scFv antibodies makes it the antibody format of choice fortumor penetration and rapid clearance from the blood (Adams et al.,1995; Adams, 1998; Yokota et al., 1992). The human phage antibodylibrary can be used to develop antibodies suitable for clinical trials.These human scFv antibodies have entered clinical trials (Hoogenboom &Winter, 1992). The human phage antibody library can be used to developantibodies suitable for clinical trials]. Anti-melanoma antibodies havebeen developed using these phage libraries (Cai & Garen, 1995), as wellas antibodies to antigens found in ovarian carcinoma (Figini et al.,1998).

Using a phage-displayed approach for the production of antibodies, scFvantibody clones that bind to a radiation-inducible target are identifiedas disclosed herein. Fv regions are sequenced and bivalent functionalreagents are designed and tested, for example using an assay asdisclosed herein. Thus, an exemplary, but non-limiting, source for anantibody, or derivative or fragment thereof, is a recombinantphage-displayed antibody library.

The recombinant phage can comprise antibody encoding nucleic acidsisolated from any suitable vertebrate species, including in alternativeembodiments mammalian species such as human, mouse, and rat. Thus, inone embodiment the recombinant phage encode an antibody wherein both thevariable and constant regions are encoded by nucleic acids isolated fromthe same species (for example, human, mouse, or rat). In anotherembodiment, the recombinant phage encode chimeric antibodies, whereinthe phrase “chimeric antibodies” (and grammatical variations thereof)refers to antibodies having variable and constant domain regions thatare derived from different species. For example, in one embodiment thechimeric antibodies are antibodies having murine variable domains andhuman constant domains.

The scFv antibodies of the presently disclosed subject matter alsoinclude humanized scFv antibodies. Humanized forms of non-human (forexample, murine) scFv antibodies are chimeric scFv antibodies thatcontain minimal sequence derived from non-human immunoglobulins.Humanized scFv antibodies include human scFvs in which residues from acomplementarity-determining region (CDR) are encoded by a nucleic acidencoding a CDR of a non-human species such as mouse, having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988;Presta, 1992). Thus, as used herein, the term “humanized” encompasseschimeric antibodies comprising a human constant region, including thoseantibodies wherein all of the residues are encoded by a human nucleicacid (see for example Shalaby et al., 1992; Mocikat et al., 1994).

B. In Vivo Screening for Ligands that Bind Irradiated Tumors

The presently disclosed subject matter provides a method for in vivoscreening for ligands that bind irradiated tumors. As used herein, theterm “in vivo screening” refers to a method of screening a library forselection of a ligand that homes to an irradiated tumor or thevasculature associated with an irradiated tumor.

The term “in vivo”, as used herein to describe methods of screening orligand selection, refers to contacting of one or more ligands toendogenous candidate target molecules, wherein the candidate targetmolecules are naturally present in a subject, or are present in a tumorin, or a tumor biopsy from, a subject, whether naturally occurring toexperimentally induced, and the contacting occurs in the subject or inthe biopsied tumor. By contrast, “in vitro” screening refers tocontacting a library of candidate ligands with one or more isolated orrecombinantly produced target molecules.

Thus, a method for screening as disclosed herein includes the steps of(a) contacting a cell with a first solution, the first solutioncomprising a plurality of phage-displayed antibodies; (b) isolating asecond solution, the second solution comprising those phage-displayedantibodies that do not bind to the cell; (c) removing anyphage-displayed antibodies bound to the cell; (d) treating the cell withradiation, wherein the treating results in a radiation-inducibleneoantigen being present on the cell; (e) contacting the cell with thesecond solution; and (f) detecting binding of a phage-displayed antibodyto the radiation-inducible neoantigen on the cell. In one embodiment, aphage-displayed antibody is a single chain fragment variable (scFv)antibody. In another embodiment, a phage-displayed antibody is an Fabantibody,

The term “administering to a subject”, when used to describe provisionof a library of molecules, is used in its broadest sense to mean thatthe library is delivered to the irradiated tumor. For example, a librarycan be provided to the circulation of the subject by injection orcannulization such that the molecules can pass through the tumor.

Alternatively or in addition, a library can be administered to anisolated tumor or tumor biopsy. Thus, a method for in vivo screening canalso comprise: (a) exposing a tumor and a control tissue to ionizingradiation; (b) administering to the tumor and to the control tissue alibrary of diverse molecules; (c) detecting one or more molecules of thelibrary that bind to the tumor and that substantially lack binding tothe control tissue, whereby a molecule that binds an irradiated tumor isidentified.

The in vivo screening methods of the presently disclosed subject mattercan further comprise administering the library to isolated tumor cellsor to isolated proteins prior to administering the library to a subjector to a tumor. For example, in vitro screening methods can be performedto select ligands that bind to particular tumor neoantigens, followed byperformance of the in vivo screening methods as disclosed herein.

In one embodiment of the presently disclosed subject matter, theradiation treatment comprises administration of less than about 2 Grays(Gy) ionizing radiation. In another embodiment, the radiation treatmentcomprises at least about 2 Gy ionizing radiation, optionally about 2 Gyto about 3 Gy ionizing radiation, or about 2 Gy to about 6 Gy ionizingradiation. In an alternative embodiment, radiation treatment comprisesabout 10 Gy to about 20 Gy ionizing radiation.

The methods of the presently disclosed subject matter can be performedusing any tumor-bearing subject or any subject suspected of having atumor. In one embodiment a subject is a warm-blooded vertebrate, inanother embodiment a mammal, and in still another embodiment a human.

In one embodiment of the presently disclosed subject matter, a libraryis administered to a tumor-bearing human subject following irradiationof the tumor. Methods and appropriate doses for administration of alibrary to a human subject are described in PCT InternationalPublication No. WO 01/09611.

In another embodiment, a tumor is experimentally induced in a subject(for example, a mouse), the tumor comprising a human cell or cell line.A library is then administered to the tumor-bearing subject either priorto or subsequent to irradiation of the tumor, as described in Example16.

Example 5 describes a representative procedure for in vivo screening ofphage-displayed peptide ligands that bind to irradiated tumor vessels inaccordance with the presently disclosed subject matter. Briefly, peptidebinding was studied in tumor blood vessels of 2 distinct tumor models:(1) GL261 glioma, and (2) Lewis lung carcinoma. Tumors were irradiatedwith 3 Gy to facilitate identification of peptide sequences that bindtumors exposed to a minimal dose of ionizing radiation. Phage wereadministered by tail vein injection into tumor bearing mice followingirradiation. Phage were recovered from the tumor thereafter. Followingmultiple rounds of sequential in vivo binding to irradiated tumors,phage were recovered and individual phage were randomly picked andsequenced.

Example 9 describes a representative procedure for in vivo screening ofphage-displayed ligands comprising single chain antibodies. The libraryused for in vivo screening was a biased library in that a pool ofantibody ligands that bind to radiation-inducible antigens werepre-selected in vitro. However, a library need not be pre-selected invitro to be used in the methods disclosed herein.

C. Recovery of Targeting Ligands

Methods for identifying targeting ligands that bind an irradiated tumorare selected based on one or more characteristics common to themolecules present in the library. For example, mass spectrometry and/orgas chromatography can be used to resolve molecules that home to anirradiated tumor. Thus, where a library comprises diverse moleculesbased generally on the structure of an organic molecule, determining thepresence of a parent peak for the particular molecule can identify aligand that binds a radiation-inducible target molecule. The use ofmatrix-assisted laser desorption/ionization coupled mass spectrometry(MALDI-MS) to identify antibody ligands that bind to radiation-inducibleneoantigens is described in more detail herein below.

If desired, a ligand can be linked to a tag, which can facilitaterecovery or identification of the molecule. A representative tag is anoligonucleotide or a small molecule such as biotin. See e.g., Brenner &Lerner, 1992 and U.S. Pat. No. 6,068,829. Alternatively, a tag cancomprise an epitope for which an antibody is commercially available.These so-called epitope tags include, but are not limited to histidinetags, c-myc tags, and the E tag encoded by, for example, pCANTAB 5E fromAmersham Biosciences (Piscataway, N.J., United States of America).Histidine tags (also called 6×His tags) can be purified, for example,using nickel-nitrilotriacetic acid (Ni-NTA) Agarose available fromQiagen Inc. (Valencia, Calif., United States of America). Epitopes fromc-myc include the sequence recognized by the monoclonal antibody 9E10,which is produced by a hybridoma (MYC 1-9E10.2) available from theAmerican Type Culture Collection (Manassas, Virgina, United States ofAmerica). See also Evan et al., 1985 and Van Ewijk et al., 1997, each ofwhich is incorporated by reference in its entirety. Polypeptides thatcomprise Amersham's E tag (GAPVPYPDPLEPR; SEQ ID NO: 25) can be purifiedusing reagents and protocols supplied by the manufacturer. In oneembodiment, an scFv antibody of the presently disclosed subject mattercomprises an epitope tag.

In addition, a tag can be a support or surface to which a molecule canbe attached. For example, a support can be a biological tag such as avirus or virus-like particle such as a bacteriophage (“phage”); abacterium; or a eukaryotic cell such as yeast, an insect cell, or amammalian cell (e.g., an endothelial progenitor cell or a leukocyte); orcan be a physical tag such as a liposome or a microbead. In oneembodiment, a support can have a diameter less than about 10 μm to about50 μm in its shortest dimension, such that the support can passrelatively unhindered through the capillary beds present in the subjectand not occlude circulation. In addition, a support can be nontoxic andbiodegradable, particularly where the subject used for in vivo screeningis not sacrificed for isolation of library molecules from the tumor.Where a molecule is linked to a support, the part of the moleculesuspected of being able to interact with a target in a cell in thesubject can be positioned so as be able to participate in theinteraction.

D. Peptide Ligands

A targeting peptide of the presently disclosed subject matter can besubject to various changes, substitutions, insertions, and deletionswhere such changes provide for certain advantages in its use. Thus, theterm “peptide” encompasses any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, conservatively substituted variants,analogs, fragments, peptoids, chemically modified peptides, and peptidemimetics. The terms “targeting peptide” or “peptide ligand” each referto a peptide as defined herein above that binds to an irradiated tumor.The modifications disclosed herein can also be applied as desired and asappropriate to antibodies, including scFv antibodies.

Peptides of the presently disclosed subject matter can comprisenaturally occurring amino acids, synthetic amino acids, geneticallyencoded amino acids, non-genetically encoded amino acids, andcombinations thereof. Peptides can include both L-form and D-form aminoacids.

Representative non-genetically encoded amino acids include but are notlimited to 2-aminoadipic acid; 3-aminoadipic acid; 6-aminopropionicacid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid);6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid;3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid;desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid;N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine;3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline;norvaline; norleucine; and ornithine.

Representative derivatized amino acids include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups can be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups canbe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine can be derivatized to form N-im-benzylhistidine.

The term “conservatively substituted variant” refers to a peptidecomprising an amino acid residue sequence substantially identical to asequence of a reference ligand of radiation inducible target in whichone or more residues have been conservatively substituted with afunctionally similar residue and which displays the targeting activityas described herein. The phrase “conservatively substituted variant”also includes peptides wherein a residue is replaced with a chemicallyderivatized residue, provided that the resulting peptide displaystargeting activity as disclosed herein.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another; the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine; the substitutionof one basic residue such as lysine, arginine or histidine for another;or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another.

Peptides of the presently disclosed subject matter also include peptidescomprising one or more additions and/or deletions or residues relativeto the sequence of a peptide whose sequence is disclosed herein, so longas the requisite targeting activity of the peptide is maintained. Theterm “fragment” refers to a peptide comprising an amino acid residuesequence shorter than that of a peptide disclosed herein.

Additional residues can also be added at either terminus of a peptidefor the purpose of providing a “linker” by which the peptides of thepresently disclosed subject matter can be conveniently affixed to alabel or solid matrix, or carrier. Amino acid residue linkers areusually at least one residue and can be 40 or more residues, more often1 to 10 residues, but do alone not constitute radiation inducible targetligands. Typical amino acid residues used for linking are tyrosine,cysteine, lysine, glutamic and aspartic acid, or the like. In addition,a peptide can be modified by terminal-NH₂ acylation (e.g., acetylation,or thioglycolic acid amidation) or by terminal-carboxylamidation (e.g.,with ammonia, methylamine, and the like terminal modifications).Terminal modifications are useful, as is well known, to reducesusceptibility by proteinase digestion, and therefore serve to prolonghalf-life of the peptides in solutions, particularly biological fluidswhere proteases can be present.

Peptide cyclization is also a useful terminal modification because ofthe stable structures formed by cyclization and in view of thebiological activities observed for such cyclic peptides as describedherein. An exemplary method for cyclizing peptides is described bySchneider & Eberle, 1993. Typically, tertbutoxycarbonyl protectedpeptide methyl ester is dissolved in methanol and sodium hydroxidesolution are added and the admixture is reacted at 20° C. tohydrolytically remove the methyl ester protecting group. Afterevaporating the solvent, the tertbutoxycarbonyl protected peptide isextracted with ethyl acetate from acidified aqueous solvent. Thetertbutoxycarbonyl protecting group is then removed under mildly acidicconditions in dioxane cosolvent. The unprotected linear peptide withfree amino and carboxyl termini so obtained is converted to itscorresponding cyclic peptide by reacting a dilute solution of the linearpeptide, in a mixture of dichloromethane and dimethylformamide, withdicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole andN-methylmorpholine. The resultant cyclic peptide is then purified bychromatography.

The term “peptoid” as used herein refers to a peptide wherein one ormore of the peptide bonds are replaced by pseudopeptide bonds includingbut not limited to a carba bond (CH₂—CH₂), a depsi bond (CO—O), ahydroxyethylene bond (CHOH—CH₂), a ketomethylene bond (CO—CH₂), amethylene-oxy bond (CH₂—O), a reduced bond (CH₂—NH), a thiomethylenebond (CH₂—S), a thiopeptide bond (CS—NH), and an N-modified bond(—NRCO—). See e.g. Corringer et al., 1993; Garbay-Jaureguiberry et al.,1992; Tung et al., 1992; Urge et al., 1992; Pavone et al., 1993.

Peptides of the presently disclosed subject matter, including peptoids,can be synthesized by any of the techniques that are known to thoseskilled in the art of peptide synthesis. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, can be used for reasonsof purity, antigenic specificity, freedom from undesired side products,ease of production, and the like. A summary of representative techniquescan be found in Stewart & Young, 1969; Merrifield, 1969; Fields & Noble,1990; and Bodanszky, 1993. Solid phase synthesis techniques can be foundin Andersson et al., 2000, references cited therein, and in U.S. Pat.Nos. 6,015,561; 6,015,881; 6,031,071; and 4,244,946. Peptide synthesisin solution is described by Schröder & Lübke, 1965. Appropriateprotective groups usable in such synthesis are described in the abovetexts and in McOmie, 1973. Peptides that include naturally occurringamino acids can also be produced using recombinant DNA technology. Inaddition, peptides comprising a specified amino acid sequence can bepurchased from commercial sources (e.g., Biopeptide Co., LLC of SanDiego, Calif., United States of America and PeptidoGenics of Livermore,Calif., United States of America).

The term “peptide mimetic” as used herein refers to a ligand that mimicsthe biological activity of a reference peptide, by substantiallyduplicating the targeting activity of the reference peptide, but it isnot a peptide or peptoid. In one embodiment, a peptide mimetic has amolecular weight of less than about 700 daltons.

A peptide mimetic can be designed by: (a) identifying the pharmacophoricgroups responsible for the targeting activity of a peptide; (b)determining the spatial arrangements of the pharmacophoric groups in theactive conformation of the peptide; and (c) selecting a pharmaceuticallyacceptable template upon which to mount the pharmacophoric groups in amanner that allows them to retain their spatial arrangement in theactive conformation of the peptide. For identification of pharmacophoricgroups responsible for targeting activity, mutant variants of thepeptide can be prepared and assayed for targeting activity.Alternatively or in addition, the three-dimensional structure of acomplex of the peptide and its target molecule can be examined forevidence of interactions, for example the fit of a peptide side chaininto a cleft of the target molecule, potential sites for hydrogenbonding, etc. The spatial arrangements of the pharmacophoric groups canbe determined by NMR spectroscopy or X-ray diffraction studies. Aninitial three-dimensional model can be refined by energy minimizationand molecular dynamics simulation. A template for modeling can beselected by reference to a template database and will typically allowthe mounting of 2-8 pharmacophores. A peptide mimetic is identifiedwherein addition of the pharmacophoric groups to the template maintainstheir spatial arrangement as in the peptide.

A peptide mimetic can also be identified by assigning a hashed bitmapstructural fingerprint to the peptide based on its chemical structure,and determining the similarity of that fingerprint to that of eachcompound in a broad chemical database. The fingerprints can bedetermined using fingerprinting software commercially distributed forthat purpose by Daylight Chemical Information Systems, Inc. (MissionViejo, Calif.) according to the vendor's instructions. Representativedatabases include but are not limited to SPREI'95 (InfoChem GmbH ofMunchen, Germany), Index Chemicus (ISI of Philadelphia, Pa., UnitedStates of America), World Drug Index (Derwent of London, UnitedKingdom), TSCA93 (United States Envrionmental Protection Agency),MedChem (Biobyte of Claremont, Calif., United States of America),Maybridge Organic Chemical Catalog (Maybridge of Cornwall, England),Available Chemicals Directory (MDL Information Systems of San Leandro,Calif., United States of America), NC196 (United States National CancerInstitute), Asinex Catalog of Organic Compounds (Asinex Ltd. of Moscow,Russia), and NP (InterBioScreen Ltd. of Moscow, Russia). A peptidemimetic of a reference peptide is selected as comprising a fingerprintwith a similarity (Tanamoto coefficient) of at least 0.85 relative tothe fingerprint of the reference peptide. Such peptide mimetics can betested for bonding to an irradiated tumor using the methods disclosedherein.

Additional techniques for the design and preparation of peptide mimeticscan be found in U.S. Pat. Nos. 5,811,392; 5,811,512; 5,578,629;5,817,879; 5,817,757; and 5,811,515.

Any peptide or peptide mimetic of the presently disclosed subject mattercan be used in the form of a pharmaceutically acceptable salt. Suitableacids which are capable of the peptides with the peptides of thepresently disclosed subject matter include inorganic acids such astrifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid,perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoricacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of thepresently disclosed subject matter include inorganic bases such assodium hydroxide, ammonium hydroxide, potassium hydroxide and the like;and organic bases such as mono-di- and tri-alkyl and aryl amines (e.g.triethylamine, diisopropyl amine, methyl amine, dimethyl amine and thelike), and optionally substituted ethanolamines (e.g. ethanolamine,diethanolamine and the like).

E. Antibody Ligands

An targeting antibody or the presently disclosed subject matter can beidentified by the in vivo screening methods disclosed herein. In oneembodiment, an antibody targeting ligand comprises: (a) a polypeptidecomprising an amino acid sequence of SEQ ID NO: 18, 20, 22, or 24; (b) apolypeptide substantially identical to SEQ ID NO: 18, 20, 22, or 24; (c)a polypeptide encoded by SEQ ID NO: 17, 19, 21, or 23; or (d) apolypeptide encoded by a polynucleotide substantially identical to SEQID NO: 17, 19, 21, or 23. Thus, the presently disclosed subject matteralso provides an isolated nucleic acid that encodes an antibodytargeting ligand comprising: (a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 17, 19, 21, or 23; or (b) a nucleicacid molecule substantially identical to SEQ ID NO: 17, 19, 21, or 23.

When phage-displayed antibodies bind to an antigen, they can beaffinity-purified using the antigen. These affinity-purified phage canthen be used to infect and introduce the antibody gene back into E.coli. The E. coli can then be grown and induced to express a soluble,non-phage-displayed, antigen-specific recombinant antibody. Phagedisplay technology is especially useful for producing antibodies toantigens that are either poorly immunogenic or readily degraded and forwhich monoclonal and/or polyclonal antibodies are difficult to obtain.P-selectin, like α_(2b)β₃, is a high priority radiation-inducibleneoantigen because it is not accessible to antibodies orimmunoconjugates until after irradiation of tumor vasculature. PhagescFv antibodies have been developed to these proteins by use ofphage-displayed antibody library containing 2×10⁹ members. Negativeselection of phage can be first performed on a control tissue, forexample untreated vascular endothelium. This can eliminate antibodiesthat nonspecifically bind to, for example, unirradiated endothelialcells. Unbound phage can then be recovered and incubated with purifiedradiation-inducible neoantigen, for example, P-selectin or α_(2b)β₃integrin. High affinity phage can then be recovered, for example by useof washing at pH 1.

The term “isolated”, as used in the context of a nucleic acid orpolypeptide, indicates that the nucleic acid or polypeptide exists apartfrom its native environment and is not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form or can exist ina non-native environment such as a transgenic host cell. In oneembodiment of the presently disclosed subject matter, “isolated” refersto the purification of an scFv antibody from a target tissue to which ithas bound.

Nucleic Acids Encoding Targeting Antibodies. The terms “nucleic acidmolecule” or “nucleic acid” each refer to deoxyribonucleotides orribonucleotides and polymers thereof in single-stranded ordouble-stranded. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar properties as the reference natural nucleic acid. The terms“nucleic acid molecule” or “nucleic acid” can also be used in place of“gene”, “cDNA”, or “mRNA”. Nucleic acids can be synthesized, or can bederived from any biological source, including any organism.

The term “substantially identical”, as used herein to describe a degreeof similarity between nucleotide sequences, refers to two or moresequences that have in one embodiment at least about least 60%, inanother embodiment at least about 70%, in another embodiment at leastabout 80%, in another embodiment about 90% to about 99%, in anotherembodiment about 95% to about 99%, and in still another embodiment about99% nucleotide identity, as measured using one of the following sequencecomparison algorithms (described herein below) or by visual inspection.The substantial identity exists in one embodiment in nucleotidesequences of at least about 100 residues, in another embodiment innucleotide sequences of at least about 150 residues, and in stillanother embodiment in nucleotide sequences comprising a full lengthcoding sequence.

Thus, substantially identical sequences can comprise mutagenizedsequences, including sequences comprising silent mutations, or variablysynthesized sequences. A mutation or variant sequence can comprise asingle base change.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules specifically or substantiallyhybridize to each other under stringent conditions. In the context ofnucleic acid hybridization, two nucleic acid sequences being comparedcan be designated a “probe” and a “target”. A “probe” is a referencenucleic acid molecule, and a “target” is a test nucleic acid molecule,often found within a heterogeneous population of nucleic acid molecules.A “target sequence” is synonymous with a “test sequence”.

An exemplary nucleotide sequence employed for hybridization studies orassays includes probe sequences that are complementary to or mimic atleast an about 14 to 40 nucleotide sequence of a nucleic acid moleculeof the presently disclosed subject matter. For this purpose, a probecomprises a region of the nucleic acid molecule other than a sequenceencoding a common immunoglobulin region. Thus, a probe can comprise asequence encoding a domain of the antibody that comprises anantigen-binding site. In one embodiment, probes comprise 14 to 20nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100,200, 300 nucleotides or up to the full length of a region of SEQ ID NO:17, 19, 21, or 23 that encodes an antigen binding site. Such fragmentscan be readily prepared by, for example, chemical synthesis of thefragment, by application of nucleic acid amplification technology, or byintroducing selected sequences into recombinant vectors for recombinantproduction.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern blot analysis are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, 1993. Generally, highly stringent hybridization andwash conditions are selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Typically, under “stringent conditions” a probe willhybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.1×SSC at 65° C.An example of stringent wash conditions is 15 minutes in 0.2×SSC bufferat 65° C. See Sambrook & Russell, 2001 for a description of SSC buffer.Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of medium stringency washconditions for a duplex of more than about 100 nucleotides, is 15minutes in 1×SSC at 45° C. An example of low stringency wash for aduplex of more than about 100 nucleotides, is 15 minutes in 4× to 6×SSCat 40° C. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1MNa⁺ ion, typically about 0.01 to 1M Na⁺ ion concentration (or othersalts) at pH 7.0-8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2-fold (or higher) than that observed for an unrelated probe inthe particular hybridization assay indicates detection of a specifichybridization.

The following are examples of hybridization and wash conditions that canbe used to identify nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the presently disclosedsubject matter. In one embodiment, a probe nucleotide sequencehybridizes to a target nucleotide sequence in 7% sodium dodecyl sulfate(SDS), 0.5M NaPO₄, 1 mM ethylenediamine tetraacetic acid (EDTA) at 50°C. followed by washing in 2×SSC, 0.1% SDS at 50° C. In one embodiment, aprobe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS),0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 1×SSC, 0.1% SDSat 50° C. In another embodiment, a probe and target sequence hybridizein 7% sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C.followed by washing in 0.5×SSC, 0.1% SDS at 50° C. In anotherembodiment, a probe and target sequence hybridize in 7% sodium dodecylsulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in0.1×SSC, 0.1% SDS at 50° C. In yet another embodiment, a probe andtarget sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5MNaPO₄, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at65° C.

A further indication that two nucleic acid sequences are substantiallyidentical is that proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. These terms are definedfurther herein below. Nucleic acid molecules that do not hybridize toeach other under stringent conditions are still substantially identicalif the corresponding proteins are substantially identical. This canoccur, for example, when two nucleotide sequences are significantlydegenerate as permitted by the genetic code.

The term “conservatively substituted variants” refers to nucleic acidsequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues. See Batzer et al., 1991;Ohtsuka et al., 1985; Rossolini et al., 1994.

The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein above, or a primer. The term“primer” as used herein refers to a contiguous sequence comprising inone embodiment about 8 or more deoxyribonucleotides or ribonucleotides,in another embodiment about 10-20 nucleotides, and in still anotherembodiment about 20-30 nucleotides of a selected nucleic acid molecule.The primers of the presently disclosed subject matter encompassoligonucleotides of sufficient length and appropriate sequence so as toprovide initiation of polymerization on a nucleic acid molecule of thepresently disclosed subject matter.

The term “elongated sequence” refers to an addition of nucleotides (orother analogous molecules) incorporated into the nucleic acid. Forexample, a polymerase (e.g., a DNA polymerase) can add sequences at the3′ terminus of the nucleic acid molecule. In addition, the nucleotidesequence can be combined with other DNA sequences, such as promoters,promoter regions, enhancers, polyadenylation signals, intronicsequences, additional restriction enzyme sites, multiple cloning sites,and other coding segments.

Nucleic acids of the presently disclosed subject matter can be cloned,synthesized, recombinantly altered, mutagenized, or combinationsthereof. Standard recombinant DNA and molecular cloning techniques usedto isolate nucleic acids are known in the art. Site-specific mutagenesisto create base pair changes, deletions, or small insertions are alsoknown in the art. See e.g., Sambrook & Russell, 2001; Silhavy et al.,1984; Glover & Hames, 1995; Ausubel, 1995.

Single Chain Antibody Polypeptides. As used herein, the phrase“substantially identical” refers to a nucleic acid sequence having inone embodiment at least about 45%, in another embodiment at least about50%, in another embodiment at least about 60%, in another embodiment atleast about 70%, in another embodiment at least about 80%, in anotherembodiment at least about 90%, in another embodiment at least about 95%,and in still another embodiment at least about 99% sequence identity,when compared over the full length of one of SEQ ID NO: 17, 19, 21, and23. Methods for determining percent identity are defined herein below.

Substantially identical polypeptides also encompass two or morepolypeptides sharing a conserved three-dimensional structure.Computational methods can be used to compare structural representations,and structural models can be generated and easily tuned to identifysimilarities around important active sites or ligand binding sites. SeeSaqi et al., 1999; Barton, 1998; Henikoff et al., 2000; Huang et al.,2000.

Substantially identical proteins also include proteins comprising anamino acid sequence comprising amino acids that are functionallyequivalent to amino acids of SEQ ID NOs: 18, 20, 22, or 24. The term“functionally equivalent” in the context of amino acid sequences isknown in the art and is based on the relative similarity of the aminoacid side-chain substituents. See Henikoff & Henikoff, 2000. Relevantfactors for consideration include side-chain hydrophobicity,hydrophilicity, charge, and size. For example, arginine, lysine, andhistidine are all positively charged residues; that alanine, glycine,and serine are all of similar size; and that phenylalanine, tryptophan,and tyrosine all have a generally similar shape. By this analysis,described further herein below, arginine, lysine, and histidine;alanine, glycine, and serine; and phenylalanine, tryptophan, andtyrosine; are defined herein as biologically functional equivalents.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982). It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In one embodiment,amino acids for which the hydropathic indices are within ±2 of theoriginal value are substituted for each other. In another embodiment,amino acids for which the hydropathic indices are within ±1 of theoriginal value are substituted for each other. And in still anotherembodiment, amino acids for which the hydropathic indices are within±0.5 of the original value are substituted for each other in makingchanges based upon similar hydropathicity values.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 describes that the greatest local average hydrophilicityof a protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity and antigenicity, e.g., with abiological property of the protein. It is understood that an amino acidcan be substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In one embodiment, amino acids for which the hydrophilic indices arewithin ±2 of the original value are substituted for each other. Inanother embodiment, amino acids for which the hydrophilic indices arewithin ±1 of the original value are substituted for each other. And instill another embodiment, amino acids for which the hydrophilic indicesare within ±0.5 of the original value are substituted for each other inmaking changes based upon similar hydropathicity values.

The term “substantially identical” also encompasses polypeptides thatare biologically functional equivalents. The term “functional”, as usedherein to describe polypeptides comprising antibody targeting ligands,refers two or more antibodies that are immunoreactive with a sameradiation-inducible target molecule. In one embodiment, the two or moreantibodies specifically bind a same target molecule and substantiallylack binding to a control antigen.

The term “specifically binds”, when used to describe binding of anantibody to a target molecule, refers to binding to a target molecule ina heterogeneous mixture of other polypeptides.

The phases “substantially lack binding” or “substantially no binding”,as used herein to describe binding of an antibody to a controlpolypeptide or sample, refers to a level of binding that encompassesnon-specific or background binding, but does not include specificbinding.

Techniques for detecting antibody-target molecule complexes are known inthe art and include but are not limited to centrifugation, affinitychromatography and other immunochemical methods. In one embodiment, anantibody-target molecule complex can be detected followingadministration of an antibody to a subject as described in Examples 6and 7. In another embodiment, an antibody-target molecule complex can bedetected in vivo by performing radiation-guided drug delivery, whereinthe drug comprises a targeting antibody of SEQ ID NO: 18, 20, 22, or 24and a detectable label, as described in Examples 1 and 2. See alsoManson, 1992; Law, 1996.

The presently disclosed subject matter also provides functionalfragments of a antibody targeting polypeptide. Such functional portionneed not comprise all or substantially all of the amino acid sequence ofSEQ ID NO: 18, 20, 22, or 24.

The presently disclosed subject matter also includes functionalpolypeptide sequences that are longer sequences than that of SEQ ID NO:18, 20, 22, or 24. For example, one or more amino acids can be added tothe N-terminus or C-terminus of a antibody targeting ligand. Methods ofpreparing such proteins are known in the art.

Isolated polypeptides and recombinantly produced polypeptides can bepurified and characterized using a variety of standard techniques thatare known to the skilled artisan. See e.g., Schröder & Lübke, 1965;Schneider & Eberle, 1993; Bodanszky, 1993; Ausubel, 1995.

Nucleotide and Amino Acid Sequence Comparisons. The terms “identical” orpercent “identity” in the context of two or more nucleotide orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the sequence comparisonalgorithms disclosed herein or by visual inspection.

The term “substantially identical” in regards to a nucleotide orpolypeptide sequence means that a particular sequence varies from thesequence of a naturally occurring sequence by one or more deletions,substitutions, or additions, the net effect of which is to retainbiological activity of a gene, gene product, or sequence of interest.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer program, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are selected. The sequencecomparison algorithm then calculates the percent sequence identity forthe designated test sequence(s) relative to the reference sequence,based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm disclosed in Smith & Waterman, 1981, by thehomology alignment algorithm disclosed in Needleman & Wunsch, 1970, bythe search for similarity method disclose din Pearson & Lipman, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG® WISCONSIN PACKAGE™, available from Accelrys Inc.,San Diego, Calif., United States of America), or by visual inspection.See generally, Ausubel, 1995.

A representative algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., 1990. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W)=11,an expectation (E)=10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix. See Henikoff & Henikoff, 1992.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See e.g., Karlin & Altschul, 1993. One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1 in one embodiment, less than about0.01 in another embodiment, and less than about 0.001 in still anotherembodiment.

F. Immunoconjugates

The presently disclosed subject matter also provides immunoconjugatecompositions comprising the antibodies and antibody fragments disclosedherein. In one embodiment, the antibody fragment is a humanized scFvantibody. In another embodiment, the antibody fragment is an scFvantibody comprising an amino acid sequence set forth in one of SEQ IDNOs: 18, 20, 22, or 24, or encoded by a nucleic acid comprising SEQ IDNOs: 17, 19, 21, or 23.

Immunoconjugates compositions of the presently disclosed subject mattercan be monovalent (i.e. they comprise an antibody that binds to only oneepitope present on a radiation-inducible neoantigen) or polyvalent. Asused herein, a “polyvalent immunoconjugate composition” refers to animmunoconjugate composition that comprises at least two differentligands (for example, scFv antibodies that bind to radiation-inducibleneoantigens) that bind to at least two different targets, at least oneof which is a radiation-inducible neoantigen. Thus, in one embodiment apolyvalent immunoconjugate composition comprises a plurality of singlechain fragment variable (scFv) antibodies, human Fab antibodies, orcombinations thereof, wherein the plurality of antibodies or antibodyfragments bind to a plurality of different epitopes, and wherein atleast one of the epitopes is present on a radiation-inducibleneoantigen. In one embodiment, at least one of the plurality ofdifferent epitopes is present on a vascular endothelial cell.

An exemplary polyvalent immunoconjugate is depicted in FIG. 1. As shownin FIG. 1, Antibody 1 binds to an epitope present on endothelium (forexample, tumor endothelium), and Antibody 2 binds to an antigen presenton vascular endothelium. One or both of the epitopes to which Antibody 1and Antibody 2 bind can be radiation-inducible neoantigens. This Figuredepicts the epitopes to which Antibodies 1 and 2 bind as beingdifferent, thus the immunoconjugate is a polyvalent immunoconjugate.However, if Antibody 1 and Antibody 2 bind to the same epitope presenton a radiation-inducible neoantigen, the immunoconjugate would bemonovalent.

In accordance with the presently disclosed subject matter,immunoconjugate compositions can be used to deliver therapeutic agentsto target tissues. Such therapeutic agents include, but are not limitedto viruses, radionuclides, cytotoxins, therapeutic genes, andchemotherapeutic agents.

Also in accordance with the presently disclosed subject matter, animmunoconjugate composition, the immunoconjugate composition can furthercomprise a detectable label. In one embodiment, the detectable label isdetectable in vivo. In this embodiment, the detectable label comprises alabel that can be detected using magnetic resonance imaging,scintigraphic imaging, ultrasound, or fluorescence. An exemplarydetectable label that can be used for detection in vivo is aradionuclide label, for example ¹³¹I or ^(99m)Tc.

In one embodiment, prioritization and quantification combinations ofantibodies employ fluorescent “bar codes”. Fluorescent core/shellsemiconductor nanocrystals, or “quantum dots”, are a new tool forfluorescent imaging in biology (Bruchez et al., 1998; Chan & Nie, 1998).The emission wavelength is precisely tuned by the size of thenanocrystal, which typically range in diameter from 1 nm to 10 nm(corresponding to blue through red emission wavelengths for a CdSe/ZnScore/shell). This small size and appropriate organic surfacemodification make the nanocrystals readily biocompatible (Dubertret etal., 2002). Unlike organic fluorescent dies, the nanocrystals havenarrow, gaussian emission spectra, enabling the visualization of severalreceptors or cellular components simultaneously. As the absorptionspectrum is continuous above the first excitation feature, all sizes,and hence all colors, can be excited with a single excitation source.Finally the nanocrystals photobleach on a timescale of hours, as opposedto minutes.

Ligands (for example, antibodies or antibody fragments that bind toradiation-inducible neoantigens) can be bound to the surface of thenanocrystal in order to image targets to which the ligands bind. Forexample, a ligand can be conjugated to a mercaptoacetic acid-coated dotvia an EDC coupling, and a 5,000 GMW polyethylene glycol chain is usedto defeat non-specific binding. Quantum dots can therefore be used toefforts to study the binding of immunoconjugates to targets.

IV. Prioritizing the Binding of scFv Antibodies

The presently disclosed subject matter also provides a method forprioritizing the binding of a plurality of phage-displayed antibodies toa target tissue in a subject, the method comprising: (a) providing aplurality of phage-displayed antibodies that bind to the target, whereinthe plurality of phage-displayed antibodies comprise at least twodifferent phage-displayed antibodies that bind a radiation-inducibleneoantigen within the target tissue, and wherein the at least twodifferent phage-displayed antibodies are distinguishable from eachother; (b) irradiating the target tissue, whereby theradiation-inducible neoantigens are expressed within the target tissue;(c) administering the plurality of phage-displayed antibodies to thesubject under conditions sufficient to allow the plurality ofphage-displayed antibodies to bind to the radiation-inducibleneoantigens in the target tissue; (d) isolating a portion of the targettissue from the subject, wherein the portion comprises theradiation-inducible neoantigens to which the plurality ofphage-displayed antibodies bind; (e) identifying the at least twodifferent phage-displayed antibodies in the portion of the targettissue; (f) comparing a relative selectivity and an affinity for theradiation-inducible neoantigens of the at least two differentphage-displayed antibodies identified in step (e) in the irradiatedtarget tissue; and (g) assigning a priority to the at least twodifferent phage-displayed antibodies based on the comparing of step (f).In one embodiment, the phage-displayed antibodies are single chainfragment variable (scFv) antibodies. In another embodiment, thephage-displayed antibodies are Fab antibodies.

As used herein, the term “prioritizing” refers to a qualitative and/orquantitative evaluation of the potential usefulness of a given antibodyfor use in the disclosed methods. For example, as described herein,parameters that can be considered in choosing antibodies (for example,scFv or Fab antibodies) for inclusion in an immunoconjugate include, butare not limited to, the affinity and the specificity of the binding ofthe antibody to a radiation-inducible neoantigen. In other words,antibodies can be evaluated (i.e. prioritized) based on their affinitiesand specificities for binding to radiation-inducible neoantigens presentin target tissues. The prioritization can include, for example,measuring the fraction of administered antibody that binds to the targettissue versus a control tissue (for example, a non-neoplastic tissue(for example, a normal cell) or non-irradiated vascular endothelium).Thus, an antibody that binds to a radiation-inducible neoantigen in asubject but substantially lacks binding to non-target tissues would beexpected to have a higher priority than one that binds to aradiation-inducible neoantigen but also shows substantial binding tonormal cells present within a subject. Additional factors that can beused to prioritize antibodies include, but are not limited to prolongedkinetics, specific binding in target tissues, successful targeting aftertherapeutic doses of radiation, binding to epitopes that are accessibleto immunoconjugates, and binding to antigens that remain tethered totumor vessels.

Several sequential and complementary high throughput screens ofantibodies and antibody fragments (for example, scFv antibodies and/orhuman Fab antibodies) that bind radiation-inducible neoantigens havebeen developed: (a) phage library screen; (b) fluorometric microvolumeassay technology (FMAT; see Stadel et al., 1997 for a discussion ofFMAT); (c) BIACORE®/ELISA/westerns; and (d) MALDI-MS. In the first step,antibodies or antibody fragments that bind radiation-inducibleneoantigens are selected from a library of phage antibodies (forexample, a library of phage-displayed scFv antibodies with a complexityof 10⁹ members). Subsequently, the other screening methodologies areused to prioritize those antibodies that have the highest bindingaffinities for neoantigens.

Fluorometric microvolume assay technology (FMAT) can be used to assaythe interactions between ligands (for example, antibodies and antibodyfragments) and targets (for example, radiation-inducible neoantigens).An example of the output from the PE Biosystems FMAT™ 8100 used toprioritize several anti-P-selectin scFv antibodies is shown in FIG. 2.

Of those antibodies, several are chosen for simultaneous administrationto patients with irradiated tumors (for example, irradiated gliomas),and MALDI-MS is used to detect those antibodies that achieve optimaltumor-specific binding. Imaging mass spectrometry (MS) can be used todevelop radiation-inducible antigens and to prioritize antibodies thatbind within tumors. This technology brings new and extraordinarilypowerful capabilities to the laboratory by allowing imaging of thepattern of a specific molecular weight protein in a tumor sample. MSoffers a unique high-accuracy molecular specificity that is invaluablein understanding the molecular events surrounding the interactions oftargeting antibodies and their targets (for example, radiation-inducibleneoantigens).

MALDI-MS can be used for analyzing large numbers of samples where themolecular weight of peptides and proteins are of prime interest. Itutilizes a solid sample mounted on a stage, mixing or coating of thesample with a crystalline organic matrix, and a laser for the depositionof energy into the sample. A time-of-flight analyzer is commonly used toassign mass-to-charge (m/z) ratios to the desorbed ions. The high dutycycle of the laser/analyzer combination permits the acquisition ofsummed spectra for multiple laser shots and quick downloads so that veryhigh sample throughput is possible. The technology is particularlysensitive for peptide and protein analysis.

Profiling and imaging techniques using MALDI-MS have been developed forthe spatial analysis of peptides and proteins in biological samples,focusing on their applications to tissue sections (Caprioli et al.,1997; Stoeckli et al., 2001). An early use of MALDI-MS for imaging cellsand tissues demonstrated that signals for peptides and proteins could beobtained directly from tissues and blots of tissues. Over the past 2years, the inventors have developed imaging MS technology (FIG. 3) andhave shown that relatively large proteins can be desorbed from tissuesand blots of tissues in the molecular weight range up to about 80 kDa.Thus, MALDI-MS can be used to prioritize antibody binding in tumorsbecause the molecular weights of scFv antibodies range from about 25 toabout 31 kDa. From tumor samples, 300-500 peptide and protein peaks canbe recorded in the mass spectrum produced from a single laser ablatedarea on the sample. Further, a raster of the surface of the sample canbe performed with multiple laser spots and the mass spectrum from eachspot saved separately, resulting in the production of a data array thatcontains the relative intensity of any mass at each spot. An image ofthe sample can then be constructed at any given molecular weight,providing a molecular weight-specific map of the sample. Ahigh-resolution image of a piece of tissue might then consist of anarray of 100 by 100 laser spots with each spot being roughly circularwith a diameter of 30 μm, covering an area of 3,000 by 3,000 μm. Boththe area covered and the density of the laser spots can be covered,depending on the task to be accomplished. Commonly, individual maps canbe generated to verify the presence, molecular weight, and location ofproteins (for example, scFv antibodies) that have been selected based onpreliminary mass spectrometry scans, 2D gels, gene identification andsequencing, and other biochemical information. From a single raster of apiece of tissue, hundreds of image maps can be produced, each at adiscrete molecular weight value.

The sequential and complementary high throughput presented herein can beused to examine scFv antibodies that can be differentiated by MALDI-MSin order to perform side-by-side comparisons of antibody binding withintumors. These comparisons can be performed by immunoprecipitating taggedantibodies from tumor homogenates, followed by MALDI-MS. In oneembodiment, scFv antibodies are tagged with c-myc and 6×His, whichprovide an approach to isolating antibodies from tumor biopsy specimensbefore they are measured by MALDI-MS. In another embodiment, the scFvantibodies are tagged with an E-tag epitope tag. FIGS. 4A and 4B and 4Cdepict an E-tagged scFv antibody and a mass spectrograph of the affinitypurification of such antibodies from a cell lysate, respectively.

V. Tumor Diagnosis, Treatment, and Imaging

The presently disclosed subject matter further provides methods andcompositions for x-ray guided drug delivery to a tumor in a subject. Theterm “drug” as used herein refers to any substance having biological ordetectable activity. Thus, the term “drug” includes a pharmaceuticalagent, a diagnostic agent, or a combination thereof. The term “drug”also includes any substance that is desirably delivered to a tumor.

Thus, in one embodiment of the presently disclosed subject matter, acomposition is prepared, the composition comprising a targeting ligandas disclosed herein and a diagnostic agent. The composition can be usedfor the detection of a tumor in a subject by: (a) exposing a suspectedtumor to ionizing radiation; (b) administering to the subject atargeting ligand of the presently disclosed subject matter, wherein theligand comprises a detectable label; and (c) detecting the detectablelabel, whereby a tumor is diagnosed. Alternatively, a method fordetecting a tumor can comprise: (a) exposing a suspected tumor toionizing radiation; (b) biopsing a suspected tumor; (c) contacting atargeting ligand of the presently disclosed subject matter with thesuspected tumor in vitro, wherein the ligand comprises a detectablelabel; and (d) detecting the detectable label, whereby a tumor isdiagnosed.

A therapeutic composition of the presently disclosed subject matter cancomprise one or more targeting ligands and a therapeutic agent, suchthat the therapeutic agent can be selectively targeted to an irradiatedtumor. The one or more targeting ligands can comprise ligands havingdiverse molecular features. For example, one or more targeting ligandscan comprise both peptide and antibody targeting ligands. In oneembodiment, a therapeutic composition is an immunoconjugate. In oneembodiment, the immunoconjugate is polyvalent, meaning that it comprisesat least two targeting ligands that bind to at least two differentepitopes, at least one of which is an epitope found on aradiation-inducible neoantigen.

Optionally, a therapeutic composition can additionally comprise adetectable label, in one embodiment a label that can be detected invivo. The biodistribution of the therapeutic composition so prepared canbe monitored following administration to a subject.

Methods for preparation, labeling, and x-ray guided drug delivery usingtargeting ligands of the presently disclosed subject matter aredescribed further herein below. See also Examples 1 and 2.

A. Therapeutic Agents

The novel targeting ligands disclosed here are used to target atherapeutic agent to an irradiated tumor. Representative therapeuticagents include but are not limited to a nucleic acid (e.g., atherapeutic gene) and a small molecule. In one embodiment of thepresently disclosed subject matter, an inactive drug is administered,which is subsequently activated by irradiation (Hallahan et al., 1995b).For example, therapeutic gene expression can be regulated by aradiation-inducible promoter (Hallahan et al., 1995b).

Therapeutic Genes. Angiogenesis and suppressed immune response play acentral role in the pathogenesis of malignant disease and tumor growth,invasion, and metastasis. Thus, a representative therapeutic geneencodes a polypeptide having an ability to induce an immune responseand/or an anti-angiogenic response in vivo.

The term “immune response” is meant to refer to any response to anantigen or antigenic determinant by the immune system of a vertebratesubject. Exemplary immune responses include humoral immune responses(e.g. production of antigen-specific antibodies) and cell-mediatedimmune responses (e.g. lymphocyte proliferation),

Representative therapeutic proteins with immunostimulatory effectsinclude but are not limited to cytokines (e.g., an interleukin (IL) suchas IL2, IL4, IL7, IL12, interferons, granulocyte-macrophagecolony-stimulating factor (GM-CSF), tumor necrosis factor alpha(TNF-α)), immunomodulatory cell surface proteins (e.g., human leukocyteantigen (HLA proteins), co-stimulatory molecules, and tumor-associatedantigens. See Kirk & Mule, 2000; Mackensen et al., 1997; Walther &Stein, 1999; and references cited therein.

The term “angiogenesis” refers to the process by which new blood vesselsare formed. The term “anti-angiogenic response” and “anti-angiogenicactivity” as used herein, each refer to a biological process wherein theformation of new blood vessels is inhibited.

Representative proteins with anti-angiogenic activities that can be usedin accordance with the presently disclosed subject matter include:thrombospondin I (Kosfeld & Frazier, 1993; Tolsma et al., 1993; Dameronet al., 1994), metallospondin proteins (Carpizo & Iruela-Arispe, 2000),class I interferons (Albini et al., 2000), IL12 (Voest et al., 1995),protamine (Ingber et al., 1990), angiostatin (O'Reilly et al., 1994),laminin (Sakamoto et al., 1991), endostatin (O'Reilly et al., 1997), anda prolactin fragment (Clapp et al., 1993). In addition, severalanti-angiogenic peptides have been isolated from these proteins (Maioneet al., 1990; Eijan et al., 1991; Woltering et al., 1991).

A gene therapy construct used in accordance with the methods of thepresently disclosed subject matter can also encode a therapeutic genethat displays both immunostimulatory and anti-angiogenic activities, forexample, IL12 (see Dias et al., 1998 and references cited herein below),interferon-α(O'Byrne et al., 2000), and references cited therein), or achemokine (Nomura & Hasegawa, 2000, and references cited therein). Inaddition, a gene therapy construct can encode a gene product withimmunostimulatory activity and a gene product having anti-angiogenicactivity. See e.g. Narvaiza et al., 2000.

Additional compositions useful for cancer therapy include but are notlimited to genes encoding tumor suppressor gene products/antigens,antimetabolites, suicide gene products, and combinations thereof. SeeKirk & Mule, 2000; Mackensen et al., 1997; Walther & Stein, 1999; andreferences cited therein.

Therapeutic Compounds. In accordance with the methods of the presentlydisclosed subject matter, a therapeutic agent can also comprise acytotoxic agent, a chemotherapeutic agent, a radionuclide, or any otheranti-tumor molecule. Studies using ligand/drug conjugates havedemonstrated that a chemotherapeutic agent can be linked to a ligand toproduce a conjugate that maintains the binding specificity of the ligandand the therapeutic function of the agent. For example, doxorubicin hasbeen linked to antibodies or peptides and the ligand/doxorubicinconjugates display cytotoxic activity (Shih et al., 1994; Lau et al.,1995; Sivam et al., 1995), PCT International Publication No. WO98/10795). Similarly, other anthracyclines, including idarubicin anddaunorubocin, have been chemically conjugated to antibodies, which havefacilitated delivery of effective doses of the agents to tumors(Aboud-Pirak et al., 1989; Rowland et al., 1993). Other chemotherapeuticagents include cis-platinum (Schechter et al., 1991), methotrexate(Shawler et al., 1988; Fitzpatrick & Garnett, 1995) and mitomycin-C(Dillman et al., 1989).

In another embodiment of the presently disclosed subject matter, atherapeutic agent comprises a radionuclide. Radionuclides can beeffectively conjugated to antibodies (Hartmann et al., 1994; Buchsbaumet al., 1995), small molecule ligands (Wilbur, 1992; Fjalling et al.,1996), and peptides (Boerman et al., 2000; Krenning & de Jong, 2000;Kwekkeboom et al., 2000; Virgolini et al., 2001, and references citedtherein), such that administration of the conjugated radionuclidepromotes tumor regression. Representative therapeutic radionuclides andmethods for preparing a radionuclide-labeled agent are described furtherherein below under the heading Scintigraphic Imaging. For therapeuticmethods of the presently disclosed subject matter, exemplaryradionuclides comprise ¹³¹I and ^(99m)Tc.

Additional anti-tumor agents that can be conjugated to the targetingligands disclosed herein and used in accordance with the therapeuticmethods of the presently disclosed subject matter include but are notlimited to alkylating agents such as melphalan and chlorambucil (Smythet al., 1987; Aboud-Pirak et al., 1989; Rowland et al., 1993), vincaalkaloids such as vindesine and vinblastine (Aboud-Pirak et al., 1989;Starling et al., 1992), antimetabolites such as 5-fluorouracil,5-fluorouridine and derivatives thereof (Krauer et al., 1992; Henn etal., 1993).

Other Therapeutic Agents. In accordance with the methods of thepresently disclosed subject matter, a therapeutic agent can comprise avirus or a viral genome. In one embodiment, a therapeutic agentcomprises an oncolytic virus. In an exemplary embodiment, an oncolyticvirus comprises a naturally occurring virus that is capable of killing acell in the target tissue (for example, by lysis) when it enters such acell. Alternatively, an oncolytic virus can comprise a recombinant viralvector (for example, an adenovirus vector) that has been engineered toencode a polypeptide that, when present in a cell of the target tissuesuppresses the growth of that cell or kills it. For example, arecombinant viral vector can comprise an adenovirus vector that has beenengineered to encode one of the therapeutic genes disclosed herein,including but not limited to immunostimulatory genes, anti-angiogenicgenes, tumor suppressors, antimetabolites, suicide gene products, andcombinations thereof.

B. Preparation of a Therapeutic and/or Diagnostic Composition

The presently disclosed subject matter also provides a method forpreparing a composition for x-ray-guided drug delivery. The methodcomprises: (a) performing in vivo screening, whereby a ligand that bindsa radiation-inducible tumor molecule is identified; and (b) conjugatingthe ligand to a drug, whereby a composition for x-ray-guided drugdelivery is prepared. A drug can further comprise a drug carrier and canbe formulated in any manner suitable for administration to a subject. Inone embodiment of the presently disclosed subject matter, the methodemploys a targeting ligand identified by in vivo screening. Suchtargeting ligands can comprise, for example, peptides (for example, thepeptides disclosed in any one of SEQ ID NOs: 1-13), scFv antibodies (forexample, the scFv antibodies disclosed in any one of SEQ ID NOs: 18, 20,22, and 24), and immunoconjugates comprising the peptides and scFvantibodies (such as those disclosed in SEQ ID NOs: 1-13, 18, 20, 22, and24).

Drug Carriers. The compositions of the presently disclosed subjectmatter can further comprise a drug carrier to facilitate drugpreparation and administration. Any suitable drug delivery vehicle orcarrier can be used, including but not limited to a gene therapy vector(e.g., a viral vector or a plasmid), a microcapsule, for example amicrosphere or a nanosphere (Manome et al., 1994; Hallahan, 2001a;Saltzman & Fung, 1997), a peptide (U.S. Pat. Nos. 6,127,339 and5,574,172), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a fatty acid(U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No. 5,651,991), alipid or lipid derivative (U.S. Pat. No. 5,786,387), collagen (U.S. Pat.No. 5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No.5,688,931), a nanosuspension (U.S. Pat. No. 5,858,410), a polymericmicelle or conjugate (Goldman et al., 1997 and U.S. Pat. Nos. 4,551,482,5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome (U.S.Pat. No. 5,922,545). In one embodiment of the presently disclosedsubject matter, a drug carrier comprises a nanosphere, and in anotherembodiment a drug carrier comprises a liposome.

Conjugation of Targeting Ligands. Antibodies, peptides, or other ligandscan be coupled to drugs or drug carriers using methods known in the art,including but not limited to carbodiimide conjugation, esterification,sodium periodate oxidation followed by reductive alkylation, andglutaraldehyde crosslinking. See Goldman et al., 1997; Cheng, 1996; Neriet al., 1997; Nabel, 1997; Park et al., 1997; Pasqualini et al., 1997;Bauminger & Wilchek, 1980; U.S. Pat. No. 6,071,890; and European PatentNo. 0 439 095.

In addition, a targeting peptide or antibody can be recombinantlyexpressed. For example, a nucleotide sequence encoding a targetingpeptide or ligand can be cloned into adenovirus DNA encoding the H1 loopfiber, such that the targeting peptide or ligand is extracellularlypresented. An adenovirus vector so prepared can be used for x-ray-guideddelivery of a gene therapy construct as disclosed herein. A modifiedadenovirus vector encoding the RGD peptide was observed to transduce theendothelium in tumor blood vessels.

Formulation. A therapeutic composition, a diagnostic composition, or acombination thereof of the presently disclosed subject matter comprisesin one embodiment a pharmaceutical composition that includes apharmaceutically acceptable carrier. Suitable formulations includeaqueous and non-aqueous sterile injection solutions which can containanti-oxidants, buffers, bacteriostats, bactericidal antibiotics andsolutes which render the formulation isotonic with the bodily fluids ofthe intended recipient; and aqueous and non-aqueous sterile suspensionswhich can include suspending agents and thickening agents. Theformulations can be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier, for example water for injections, immediatelyprior to use. Some exemplary ingredients are SDS, for example in therange of 0.1 to 10 mg/ml, in one embodiment about 2.0 mg/ml; and/ormannitol or another sugar, for example in the range of 10 to 100 mg/ml,in one embodiment about 30 mg/ml; and/or phosphate-buffered saline(PBS). Any other agents conventional in the art having regard to thetype of formulation in question can be used.

The therapeutic regimens and pharmaceutical compositions of thepresently disclosed subject matter can be used with additional adjuvantsor biological response modifiers including, but not limited to, thecytokines interferon (IFN)-α, IFN-γ, IL2, IL4, IL6, tumor necrosisfactor (TNF), or other cytokine affecting immune cells.

C. Administration

Suitable methods for administration of a therapeutic composition, adiagnostic composition, or combination thereof, of the presentlydisclosed subject matter include but are not limited to intravascular,subcutaneous, or intratumoral administration. In one embodiment,intravascular administration is employed. For delivery of compositionsto pulmonary pathways, compositions can be administered as an aerosol orcoarse spray.

For therapeutic applications, a therapeutically effective amount of acomposition of the presently disclosed subject matter is administered toa subject. A “therapeutically effective amount” is an amount of thetherapeutic composition sufficient to produce a measurable biologicaltumor response (e.g., an immunostimulatory, an anti-angiogenic response,a cytotoxic response, or tumor regression). Actual dosage levels ofactive ingredients in a therapeutic composition of the presentlydisclosed subject matter can be varied so as to administer an amount ofthe active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject. The selected dosage levelwill depend upon a variety of factors including the activity of thetherapeutic composition, formulation, the route of administration,combination with other drugs or treatments, tumor size and longevity,and the physical condition and prior medical history of the subjectbeing treated. In one embodiment, a minimal dose is administered, anddose is escalated in the absence of dose-limiting toxicity.Determination and adjustment of a therapeutically effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine.

For diagnostic applications, a detectable amount of a composition of thepresently disclosed subject matter is administered to a subject. A“detectable amount”, as used herein to refer to a diagnosticcomposition, refers to a dose of such a composition that the presence ofthe composition can be determined in vivo or in vitro. A detectableamount will vary according to a variety of factors, including but notlimited to chemical features of the drug being labeled, the detectablelabel, labeling methods, the method of imaging and parameters relatedthereto, metabolism of the labeled drug in the subject, the stability ofthe label (e.g. the half-life of a radionuclide label), the time elapsedfollowing administration of the drug and/or labeled antibody prior toimaging, the route of drug administration, the physical condition andprior medical history of the subject, and the size and longevity of thetumor or suspected tumor. Thus, a detectable amount can vary and can betailored to a particular application. After study of the presentdisclosure, and in particular the Examples, it is within the skill ofone in the art to determine such a detectable amount.

D. Radiation Treatment

The disclosed targeting ligands are useful for x-ray guided drugdelivery. Targeted drug delivery to a tumor in a subject can beperformed by irradiating the tumor prior to, concurrent with, orsubsequent to administration of a composition of the presently disclosedsubject matter. In accordance with the in vivo screening methods fordiscovery of the targeting ligands, the tumor is irradiated in oneembodiment 0 hours to about 24 hours before administration of thecomposition, and in another embodiment about 4 hours to about 24 hoursbefore administration of the composition.

Low doses of radiation can be used for selective targeting using thepeptide ligands disclosed herein. In one embodiment, the dose ofradiation comprises up to about 2 Gy ionizing radiation. Higherradiation doses can also be used, especially in the case of localradiation treatment as described herein below.

Radiation can be localized to a tumor using conformal irradiation,brachytherapy, or stereotactic irradiation. The threshold dose forinductive changes can thereby be exceeded in the target tissue butavoided in surrounding normal tissues. In this case, a dose of at leastabout 2 Gy ionizing radiation can be used; in one embodiment, about 10Gy to about 20 Gy ionizing radiation is used. For treatment of a subjecthaving two or more tumors, local irradiation enables differential drugadministration and/or dose at each of the two or more tumors.Alternatively, whole body irradiation can be used, as permitted by thelow doses of radiation required for targeting of ligands disclosedherein. Radiotherapy methods suitable for use in the practice of thispresently disclosed subject matter can be found in Leibel & Phillips,1998, among other sources.

E. Monitoring Distribution In Vivo

In one embodiment of the presently disclosed subject matter, adiagnostic and/or therapeutic composition for x-ray-guided drug deliverycomprises a label that can be detected in vivo. The term “in vivo”, asused herein to describe imaging or detection methods, refer to generallynon-invasive methods such as scintigraphic methods, magnetic resonanceimaging, ultrasound, or fluorescence, each described briefly hereinbelow. The term “non-invasive methods” does not exclude methodsemploying administration of a contrast agent to facilitate in vivoimaging.

The label can be conjugated or otherwise associated with a targetingligand (e.g., any one of SEQ ID NOs: 1-13, 18, 20, 22, and 24), atherapeutic, a diagnostic agent, a drug carrier, or combinationsthereof. Following administration of the labeled composition to asubject, and after a time sufficient for binding, the biodistribution ofthe composition can be visualized. The term “time sufficient forbinding” refers to a temporal duration that permits binding of thelabeled agent to a radiation-inducible target molecule.

Scintigraphic Imaging. Scintigraphic imaging methods include SPECT(Single Photon Emission Computed Tomography), PET (Positron EmissionTomography), gamma camera imaging, and rectilinear scanning. A gammacamera and a rectilinear scanner each represent instruments that detectradioactivity in a single plane. Most SPECT systems are based on the useof one or more gamma cameras that are rotated about the subject ofanalysis, and thus integrate radioactivity in more than one dimension.PET systems comprise an array of detectors in a ring that also detectradioactivity in multiple dimensions.

A representative method for SPECT imaging is presented in Example 2.Other imaging instruments suitable for practicing the method of thepresently disclosed subject matter, and instruction for using the same,are readily available from commercial sources. Both PET and SPECTsystems are offered by ADAC (Milpitas, Calif., United States of America)and Siemens (Hoffman Estates, Illinois, United States of America).Related devices for scintigraphic imaging can also be used, such as aradio-imaging device that includes a plurality of sensors withcollimating structures having a common source focus.

When scintigraphic imaging is employed, the detectable label cancomprise a radionuclide label; in alternative embodiements, aradionuclide label selected from the group consisting of ¹⁸fluorine,⁶⁴copper, ⁶⁵copper, ⁶⁷gallium, ⁶⁸gallium, ⁷⁷bromine, ^(80m)bromine,⁹⁵ruthenium, ⁹⁷ruthenium, ¹⁰³ruthenium, ¹⁰⁶ruthenium, ^(99m)technetium,¹⁰⁷mercury, ²⁰³mercury, ¹²³iodine, ¹²⁴iodine, ¹²⁶iodine, ¹²⁶iodine,¹³¹iodine, ¹³³iodine, ¹¹¹indium, ¹¹³mindium, ^(99m)rhenium, ¹⁰⁵rhenium,¹⁰¹rhenium, ¹⁸⁶rhenium, ¹⁸⁸rhenium, ¹²¹mtellurium, ^(122m)tellurium,^(126m)tellurium, ¹⁶⁵thulium, ¹⁶⁷thulium, ¹⁶⁸thulium, and nitride oroxide forms derived there from. In one embodiment of the presentlydisclosed subject matter, the radionuclide label comprises ¹³¹ iodine or^(99m)technetium.

Methods for radionuclide-labeling of a molecule so as to be used inaccordance with the disclosed methods are known in the art. For example,a targeting molecule can be derivatized so that a radioisotope can bebound directly to it (Yoo et al., 1997). Alternatively, a linker can beadded that to enable conjugation. Representative linkers includediethylenetriamine pentaacetate (DTPA)-isothiocyanate, succinimidyl6-hydrazinium nicotinate hydrochloride (SHNH), and hexamethylpropyleneamine oxime (HMPAO) (Chattopadhyay et al., 2001; Sagiuchi et al., 2001;Dewanjee et al., 1994; U.S. Pat. No. 6,024,938). Additional methods canbe found in U.S. Pat. No. 6,080,384; Hnatowich et al., 1996; andTavitian et al., 1998.

When the labeling moiety is a radionuclide, stabilizers to prevent orminimize radiolytic damage, such as ascorbic acid, gentisic acid, orother appropriate antioxidants, can be added to the compositioncomprising the labeled targeting molecule.

Magnetic Resonance Imaging (MRI). Magnetic resonance image-basedtechniques create images based on the relative relaxation rates of waterprotons in unique chemical environments. As used herein, the term“magnetic resonance imaging” refers to magnetic source techniquesincluding convention magnetic resonance imaging, magnetization transferimaging (MTI), proton magnetic resonance spectroscopy (MRS),diffusion-weighted imaging (DWI) and functional MR imaging (fMRI). SeeRovaris et al., 2001; Pomper & Port, 2000; and references cited therein.

Contrast agents for magnetic source imaging include but are not limitedto paramagnetic or superparamagnetic ions, iron oxide particles(Weissleder et al., 1992; Shen et al., 1993), and water-soluble contrastagents. Paramagnetic and superparamagnetic ions can be selected from thegroup of metals including iron, copper, manganese, chromium, erbium,europium, dysprosium, holmium and gadolinium. Non-limiting examples ofmetals are iron, manganese and gadolinium. In one embodiment, a metal isgadolinium.

Those skilled in the art of diagnostic labeling recognize that metalions can be bound by chelating moieties, which in turn can be conjugatedto a therapeutic agent in accordance with the methods of the presentlydisclosed subject matter. For example, gadolinium ions are chelated bydiethylenetriamine pentaacetic acid (DTPA). Lanthanide ions are chelatedby tetaazacyclododocane compounds. See U.S. Pat. Nos. 5,738,837 and5,707,605. Alternatively, a contrast agent can be carried in a liposome(Schwendener, 1992).

Images derived used a magnetic source can be acquired using, forexample, a superconducting quantum interference device magnetometer(SQUID, available with instruction from Quantum Design of San Diego,Calif.). See U.S. Pat. No. 5,738,837.

Ultrasound. Ultrasound imaging can be used to obtain quantitative andstructural information of a target tissue, including a tumor.Administration of a contrast agent, such as gas microbubbles, canenhance visualization of the target tissue during an ultrasoundexamination. In one embodiment, the contrast agent can be selectivelytargeted to the target tissue of interest, for example by using apeptide for x-ray guided drug delivery as disclosed herein.Representative agents for providing microbubbles in vivo include but arenot limited to gas-filled lipophilic or lipid-based bubbles (e.g., U.S.Pat. Nos. 6,245,318, 6,231,834, 6,221,018, and 5,088,499). In addition,gas or liquid can be entrapped in porous inorganic particles thatfacilitate microbubble release upon delivery to a subject (U.S. Pat.Nos. 6,254,852 and 5,147,631).

Gases, liquids, and combinations thereof suitable for use with thepresently disclosed subject matter include air; nitrogen; oxygen; iscarbon dioxide; hydrogen; nitrous oxide; an inert gas such as helium,argon, xenon or krypton; a sulphur fluoride such as sulphurhexafluoride, disulphur decafluoride or trifluoromethylsulphurpentafluoride; selenium hexafluoride; an optionally halogenated silanesuch as tetramethylsilane; a low molecular weight hydrocarbon (e.g.containing up to 7 carbon atoms), for example an alkane such as methane,ethane, a propane, a butane or a pentane, a cycloalkane such ascyclobutane or cyclopentane, an alkene such as propene or a butene, oran alkyne such as acetylene; an ether; a ketone; an ester; a halogenatedlow molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms);or a mixture of any of the foregoing. Halogenated hydrocarbon gases canshow extended longevity, and thus can be used for some applications.Representative gases of this group include decafluorobutane,octafluorocyclobutane, decafluoroisobutane, octafluoropropane,octafluorocyclopropane, dodecafluoropentane, decafluorocyclopentane,decafluoroisopentane, perfluoropexane, perfluorocyclohexane,perfluoroisohexane, sulfur hexafluoride, and perfluorooctaines,perfluorononanes; perfluorodecanes, optionally brominated.

Attachment of targeting ligands to lipophilic bubbles can beaccomplished via chemical crosslinking agents in accordance withstandard protein-polymer or protein-lipid attachment methods (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or N-succinimidyl3-(2-pyridylthio)propionate (SPDP)). To improve targeting efficiency,large gas-filled bubbles can be coupled to a targeting ligand using aflexible spacer arm, such as a branched or linear synthetic polymer(U.S. Pat. No. 6,245,318). A targeting ligand can be attached to theporous inorganic particles by coating, adsorbing, layering, or reactingthe outside surface of the particle with the targeting ligand (U.S. Pat.No. 6,254,852).

A description of ultrasound equipment and technical methods foracquiring an ultrasound dataset can be found in Coatney, 2001; Lees,2001; and references cited therein.

Fluorescent Imaging. Non-invasive imaging methods can also comprisedetection of a fluorescent label. A drug comprising a lipophiliccomponent (therapeutic agent, diagnostic agent, vector, or drug carrier)can be labeled with any one of a variety of lipophilic dyes that aresuitable for in vivo imaging. See e.g. Fraser, 1996; Ragnarson et al.,1992; and Heredia et al., 1991. Representative labels include, but arenot limited to carbocyanine and aminostyryl dyes; in one embodiment longchain dialkyl carbocyanines (e.g., Dil, DiO, and DiD available fromMolecular Probes Inc. of Eugene, Oreg., United States of America) anddialkylaminostyryl dyes. Lipophilic fluorescent labels can beincorporated using methods known to one of skill in the art. For exampleVYBRANT™ cell labeling solutions are effective for labeling of culturedcells of other lipophilic components (Molecular Probes Inc. of Eugene,Oreg., United States of America). Preparation of liposomes comprising atargeting ligand and a Dil detectable label are described in Example 1.

A fluorescent label can also comprise sulfonated cyanine dyes, includingCy5.5 and Cy5 (available from Amersham of Arlington Heights, Ill.),IRD41 and IRD700 (available from Li-Cor, Inc. of Lincoln, Nebr.), NIR-1(available from Dejindo of Kumamoto, Japan), and LaJolla Blue (availablefrom Diatron of Miami, Fla.). See also Licha et al., 2000; Weissleder etal., 1999; and Vinogradov et al., 1996.

In addition, a fluorescent label can comprise an organic chelate derivedfrom lanthanide ions, for example fluorescent chelates of terbium andeuropium (U.S. Pat. No. 5,928,627). Such labels can be conjugated orcovalently linked to a drug as disclosed therein.

For in vivo detection of a fluorescent label, an image is created usingemission and absorbance spectra that are appropriate for the particularlabel used. The image can be visualized, for example, by diffuse opticalspectroscopy. Additional methods and imaging systems are described inU.S. Pat. Nos. 5,865,754; 6,083,486; and 6,246,901, among other places.

F. In Vitro Detection

The presently disclosed subject matter further provides methods fordiagnosing a tumor, wherein a tumor sample or biopsy is evaluated invitro. In this case, a targeting ligand of the presently disclosedsubject matter comprises a detectable label such as a fluorescent,epitope, or radioactive label, each described briefly herein below.

Fluorescence. Any detectable fluorescent dye can be used, including butnot limited to FITC (fluorescein isothiocyanate), FLUOR X™, ALEXAFLUOR®, OREGON GREEN®, TMR (tetramethylrhodamine), ROX (X-rhodamine),TEXAS RED®, BODIPY® 630/650, and Cy5 (available from Amersham PharmaciaBiotech of Piscataway, N.J. or from Molecular Probes Inc. of Eugene,Oreg.).

A fluorescent label can be detected directly using emission andabsorbance spectra that are appropriate for the particular label used.Common research equipment has been developed for in vitro detection offluorescence, including instruments available from GSI Lumonics(Watertown, Mass., United States of America) and Genetic MicroSystemsInc. (Woburn, Mass., United States of America). Most of the commercialsystems use some form of scanning technology with photomultiplier tubedetection. Criteria for consideration when analyzing fluorescent samplesare summarized by Alexay et al., 1996.

Detection of an Epitope. If an epitope label has been used, a protein orcompound that binds the epitope can be used to detect the epitope. Arepresentative epitope label is biotin, which can be detected by bindingof an avidin-conjugated fluorophore, for example avidin-FITC, asdescribed in Example 7. Alternatively, the label can be detected bybinding of an avidin-horseradish peroxidase (HRP) streptavidinconjugate, followed by colorimetric detection of an HRP enzymaticproduct. The production of a colorimetric or luminescentproduct/conjugate is measurable using a spectrophotometer orluminometer, respectively.

Autoradiographic Detection. In the case of a radioactive label (e.g.,¹³¹I or ^(99m)Tc) detection can be accomplished by conventionalautoradiography or by using a phosphorimager as is known to one of skillin the art. An exemplary autoradiographic method employs photostimulableluminescence imaging plates (Fuji Medical Systems of Stamford, Conn.).Briefly, photostimulable luminescence is the quantity of light emittedfrom irradiated phosphorous plates following stimulation with a laserduring scanning. The luminescent response of the plates is linearlyproportional to the activity (Amemiya et al., 1988; Hallahan et al.,2001b).

VI. Identification of a Radiation-Inducible Neoantigens

Targeting ligands obtained using the methods disclosed herein can beused to identify and/or isolate a target molecule that is recognized bythe targeting ligand. Representative methods include affinitychromatography, biotin trapping, and two-hybrid analysis, each describedbriefly herein below.

Affinity Chromatography. A representative method for identification of aradiation-inducible target molecule is affinity chromatography. Forexample, a targeting ligand as disclosed herein can be linked to a solidsupport such as a chromatography matrix. A sample derived from anirradiated tumor is prepared according to known methods in the art, andsuch sample is provided to the column to permit binding of a targetmolecule. The target molecule, which forms a complex with the targetingligand, is eluted from the column and collected in a substantiallyisolated form. The substantially isolated target molecule is thencharacterized using standard methods in the art. See Deutscher, 1990.

Biotin Trapping. A related method employs a biotin-labeled targetingligand such that a complex comprising the biotin-labeled targetingligand bound to a target molecule can be purified based on affinity toavidin, which is provided on a support (e.g., beads, a column). Atargeting ligand comprising a biotin label can be prepared by any one ofseveral methods, including binding of biotin maleimide[3-(N-maleimidylpropionyl)biocytin] to cysteine residues of a peptideligand (Tang & Casey, 1999), binding of biotin to a biotin acceptordomain, for example that described in K. pneumoniae oxaloacetatedecarboxylase, in the presence of biotin ligase (Julien et al., 2000),attachment of biotin amine to reduced sulfhydryl groups (U.S. Pat. No.5,168,037), and chemical introduction of a biotin group into a nucleicacid ligand, (Carninci et al., 1996). In one embodiment, abiotin-labeled targeting ligand and the unlabeled same target ligandshow substantially similar binding to a target molecule.

Two-Hybrid Analysis. As another example, targeting ligands can be usedto identify a target molecule using a two-hybrid assay, for example ayeast two-hybrid or mammalian two-hybrid assay. In one embodiment of themethod, a targeting ligand is fused to a DNA binding domain from atranscription factor (this fusion protein is called the “bait”).Representative DNA-binding domains include those derived from GAL4,LEXA, and mutant forms thereof. One or more candidate target moleculesare fused to a transactivation domain of a transcription factor (thisfusion protein is called the “prey”). Representative transactivationdomains include those derived from E. coli B42, GAL4 activation domainII, herpes simplex virus VP16, and mutant forms thereof. The fusionproteins can also include a nuclear localization signal.

The transactivation domain should be complementary to the DNA-bindingdomain, meaning that it should interact with the DNA-binding domain soas to activate transcription of a reporter gene comprising a bindingsite for the DNA-binding domain. Representative reporter genes enablegenetic selection for prototrophy (e.g. LEU2, HIS3, or LYS2 reporters)or by screening with chromogenic substrates (lacZ reporter).

The fusion proteins can be expressed from a same vector or differentvectors. The reporter gene can be expressed from a same vector as eitherfusion protein (or both proteins), or from a different vector. The bait,prey, and reporter genes are co-transfected into an assay cell, forexample a microbial cell (e.g., a bacterial or yeast cell), aninvertebrate cell (e.g., an insect cell), or a vertebrate cell (e.g., amammalian cell, including a human cell). Cells that display activity ofthe encoded reporter are indicative of a binding interaction between thepeptide and the candidate target molecule. The protein encoded by such aclone is identified using standard protocols known to one of skill inthe art.

Additional methods for yeast two-hybrid analysis can be found in Brent &Finley, 1997; Allen et al., 1995; Lecrenier et al., 1998; Yang et al.,1995; Bendixen et al., 1994; Fuller et al., 1998; Cohen et al., 1998;Kolonin & Finley, 1998; Vasavada et al., 1991; Rehrauer et al., 1996;Fields & Song, 1989.

Mass Spectroscopy. MALDI-MS can be used to identify radiation-inducibleneoantigens that are well suited for immunoconjugate-mediated drugdelivery. These include antigens that are not expressed in normalvasculature, but are inducible and tethered within tumor blood vesselsand stroma. The host components of tumors (vasculature and stroma)respond to ionizing radiation with physiologic responses that occurwithin most if not all tumors. These include responses to oxidativestress and tissue injury such as receptor and enzyme activation. Theresponse in vasculature of heterotopic tumors implanted into mice isdescribed herein.

Novel radiation-inducible neoantigens can also be identified byanalyzing the response of human head and neck squamous cell carcinoma(HNSCC) from biopsies of tumors following irradiation and characterizingthe proteomic response to irradiation within both microvasculature andstroma. For example, the response of stroma and endothelium followingirradiation of tumors can be analyzed to detect sites of apoptosis usingterminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)staining. Using this approach, it was observed that irradiated tumorendothelial respond with apoptosis which provides neoantigenic targetsfor drug delivery.

EXAMPLES

The following Examples have been included to illustrate modes of thepresently disclosed subject matter. Certain aspects of the followingExamples are described in terms of techniques and procedures found orcontemplated by the present co-inventors to work well in the practice ofthe presently disclosed subject matter. These Examples illustratestandard laboratory practices of the co-inventors. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Example 1 X-Ray Guided Delivery of Fibrinogen-Conjugated

Liposomes and Microspheres

Preparation of Radiolabeled Microspheres. Albumin microspheres (Martodamet al., 1979) were resuspended using 10 ml of sterile normal saline(0.9% NaCl). One-half milliliter of the reconstituted microsphere wasadded to a 1.5 ml conical polypropylene tube previously coated withIODO-GEN® (Pierce Biotechnology, Inc., Rockford, Ill., United States ofAmerica). To this, 11.3 mCi (418 megabecquerel (MBq)) of ¹³¹I (DuPontPharmaceuticals, Wilmington, Del., United States of America) was addedin approximately 11 μl of saline and allowed to incubate at roomtemperature for 30 minutes. Following incubation, the microspheres weretransferred to a 15 ml sterile centrifuge tube, diluted to 10 ml withnormal saline, and centrifuged at 1,500 g for seven minutes. Thesupernatant was removed and discarded. The microspheres were washed oneadditional time with 10 ml of normal saline and centrifuged. Themicrospheres were suspended in 2 ml of normal saline for injection.Final yield was 4.8 mCi (177.6 MBq) of radioiodinated microspheres in 2ml saline. Radiochemical yield was 42.4%.

Preparation of Fibrinogen-Conjugated Liposomes. The lipophilic SHreactive reagent with a long spacing arm was synthesized frommaleimide-PEG 2000-NSH ester (Prochem Chemicals, High Point, N.C.,United States of America), dioleoylphosphatidylethanolanime (DOPE,available from AVANTI® Polar Lipids, Inc., Alabaster, Ala., UnitedStates of America) and triethylamine in chloroform (1:1:1.5). Resultingmaleimide-PEG 2000-DOPE was purified by flash column. Under stirring, toa solution of fibrinogen (2 mg/ml) in 0.01 M HEPES 0.15 NaCl buffer pH7.9, containing 10 mM EDTA and 0.08% NaN₃ was added in 5-fold excess offreshly prepared Traut's reagent (2-iminothiolane hydrochloride) in thesame buffer. The reaction was allowed to proceed for 30 minutes at 0° C.

SH-fibrinogen was purified using a PD-10 desalting and buffer exchangecolumn (Amersham Pharmacia Biotech, Piscataway, N.J., United States ofAmerica). PEG 2000-PE, cholesterol, Dipalmitoyl phosphocholine (AVANTI®Polar Lipids, Inc., Alabaster, Ala., United States of America), Dil(lipid fluorescent marker available from Molecular Probes, Eugene,Oreg., United States of America), and maleimide-PEG-2000-DOPE weredissolved in chloroform and mixed at a molar ratio of 10:43:43:2:2,respectively, in a round bottom flask. The organic solvent was removedby evaporation followed by desiccation under vacuum for 2 hours.Liposomes were prepared by hydrating the dried lipid film in PBS at alipid concentration of 10 mM. The suspension was then sonicated 3×5minutes, or until the solution appeared clear, to form unilamellarliposomes of 100 nM in diameter. To conjugate thiolated fibrinogen tomaleimide containing liposomes, prepared vesicles and thiolated proteinwere mixed in 10 mm HEPES, 0.15 M NaCl and EDTA pH 6.5. The finalconcentrations for proteins and liposomes were 0.25 g/L and 2.5 mM,respectively. The peptide/liposome mixture was incubated for 18 hours atroom temperature. Vesicles were then separated from unconjugated peptideusing a sepharose 4B-CL filtration column (Amersham Pharmacia Biotech,Piscataway, N.J., United States of America).

Liposomes were fluorescently labeled with Dil fluorescent marker(Molecular Probes, Inc., Eugene, Oreg., United States of America)according to the manufacturer's instructions. Labeled liposomes wereadministered by tail vein injection to tumor bearing mice. Tumors weretreated with 4 Gy either prior to administration or after administrationof fibrinogen-liposome conjugates. Tumors were fixed and sectioned at 24hours following irradiation. Fluorescence was imaged by ultravioletmicroscopy (100×).

Image Analysis. Tumors were grown in the hind limb of nude and C57BL/6mice and irradiated with 4 Gy as described in Hallahan et al., 1995b andHallahan et al., 1998. Tumors were produced by injection of about 10⁶GL261 (glioblastoma multiforme), B16F0 (murine melanoma), or Lewis lungcarcinoma cells were injected into the hind limb(s) of mice and allowedto grow to a size of about 0.7 to 1.0 cm. ¹³¹I-fibrinogen was thenadministered by tail vein injection and gamma camera images wereobtained. Tumor bearing mice were imaged at one hour and 24 hourspost-administration of radiolabeled proteins. Planar pinhole gammacamera imaging was performed on a single-head gamma camera (HELIX® modelfrom General Electric Medical Systems, Milwaukee, Wis., United States ofAmerica) using a cone-shaped pinhole collimator with a 4-mm diameterTungsten aperture. Pinhole collimation offers the advantage of improvedphoton detection efficiency (sensitivity) and spatial resolution whencompared with conventional, parallel multi-hole collimators. Pinholeplanar imaging with a small source-aperture separation can providehigh-resolution images combined with large magnification. Each scanconsisted of a 180-second acquisition (256×256 acquisition matrix) witha 10% energy window centered on 364 keV. The source-aperture separationwas 6.0 cm.

Prior to imaging analysis in animals, a uniform ¹³¹I disk source wasimaged in order to measure the angular dependence of the pinholecollimator—gamma camera system detection efficiency with distance fromthe center of the pinhole. Angular sensitivity, normalized to 1.0 at thecenter of the pinhole, was then used to scale the mouse data in order tocorrect image counts for this geometrical effect. A calibration sourceof known ¹³¹I activity was also scanned at a 6.0 cm source-apertureseparation distance in order to measure system sensitivity along thecenter of the pinhole.

Peptide biodistribution data was assessed using two measures: (1)tumor-to-background ratio (T/B) of observed activity; and (2) tumoruptake activity in microcuries (μCi). Both types of data were obtainedusing region of interest (ROI) analysis. For both measurements an 11×11ROI was used to determine mean counts within the tumor (σ_(T)) and atfive different locations within the mouse background (σ_(B)). Thesereadings were scaled to account for geometric sensitivity and the ratioof tumor uptake to total animal uptake (R) was computed according to therelation,

$R = {\frac{\sigma_{T}}{( {\sigma_{T} + \sigma_{B}} )}.}$

Activity uptake in the tumor was then approximated by the product of theamount of activity administered into the animal multiplied by the valueobtained for R above. Tumor-background ratios were determined accordingto the general expression:

$( \frac{T}{B} ) = {\frac{\sigma_{T}}{\sigma_{B}}.}$Fibrinogen Coated Microsphere Localize to Irradiated Tumors.

Fibrinogen-coated microspheres were radiolabeled with ¹³¹I andadministered by tail vein injection into tumor bearing mice, and tumorswere irradiated with 6 Gy. The specificity of fibrinogen-coated albuminwas determined by measuring the intensity of gamma detection withinregions of interest (ROI) and well counts of tumor and other tissues. Inanimals receiving localized radiation at the tumor site, 90% of themeasured radioactivity was localized to the tumor, and 10% of theradioactivity was diffusely distributed throughout the entire animalmodel. In untreated controls, 10% of radioactive counts were localizedto the tumor (p<0.001).

During optimization studies, tumors were irradiated immediately beforeor immediately after tail vein injection. Both schedules were effectivein achieving ¹³¹I-fibrinogen-coated microsphere binding. However, tumorirradiation subsequent to microsphere administration achieved increasedtargeting specificity when compared to tumors irradiated prior tomicrosphere administration. Microspheres lacking the fibrinogen liganddid not bind irradiated tumors.

To quantify a level of binding of fibrinogen-coated microspheres inirradiated tumors, data were normalized based on background levels ofradiation. Fibrinogen-coated microspheres were 100-fold more abundant inirradiated tumors compared to non-tumor control tissues. By contrast,microspheres lacking the fibrinogen ligand were detected at similarlevels in tumor and non-tumor control tissues.

To determine whether fibrinogen-conjugated microspheres bind irradiatednon-tumor control tissues, the entire hind quarters of mice bearing hindlimb tumors were irradiated, and radiolabeled fibrinogen-coatedmicrospheres were administered immediately after irradiation. Wellcounts of all tissues were performed at 24 hours after irradiation. 90%of radioactive counts were detected in the tumor. By contrast, 2% ofradioactive counts were detected in irradiated non-tumor control tissue,demonstrating selective targeting of fibrinogen-coated microspheres toirradiated tumors.

Fibrinogen-Liposome Conjugates Localize to Irradiated Tumors.

Fibrinogen-conjugated, fluorescently labeled liposomes were administeredby tail vein into mice bearing tumors on both hind limbs. The righttumor was treated with radiation and the left tumor served as theuntreated control. Untreated control tumors showed nofibrinogen-liposome conjugate binding whereas tumors irradiatedimmediately before or immediately after tail vein injection showedfibrinogen adhesion in blood vessels. The fluorescent marker wasobserved within the vascular lumen of tumor microvasculature.

Studies using radiolabeled fibrinogen-conjugated liposomes gave similarresults. When liposomes were administered after tumor irradiation, 89%of fibrinogen-coated liposomes localized to tumors. When liposomes wereadministered immediately prior to tumor irradiation, 69% of liposomesshowed tumor localization. By contrast, in untreated controls, abackground level of 9% of fibrinogen-coated liposomes localized to thetumor.

Example 2 Clinical Trials of X-Ray-Guided Delivery Using a PeptideLigand

Ligand Preparation and Administration

Biapcitide (ACUTECT® available from Diatide, Inc., Londonderry, N.H.,United States of America) is a synthetic peptide that binds toGP-IIb/IIIa receptors on activated platelets (Hawiger et al., 1989;Hawiger & Timmons, 1992). Biapcitide was labeled with ^(99m)Tc inaccordance with a protocol provided by Diatide Inc.

Reconstituted ^(99m)Tc-labeled biapcitide was administered to patientsat a dose of 100 mcg of biapcitide radiolabeled with 10 millicuries(mCi) of ^(99m)Tc. Patients received ^(99m)Tc-labeled biapcitideintravenously immediately prior to irradiation. Patients were thentreated with 10 Gy or more. Patients underwent gamma camera imagingprior to irradiation and 24 hours following irradiation. Followingplanar image acquisition, those patients showing uptake in irradiatedtumors underwent tomographic imaging using SPECT and repeat imaging at24 hours. Patients showing no uptake on planer images during this24-hour time frame had no further imaging. Each patient had an internalcontrol, which consisted of a baseline scan immediately followingadministration of ^(99m)Tc-labeled biapcitide.

Patients were treated with X-irradiation ranging from 4 to 18 MV photonusing external beam linear accelerator at Vanderbilt University.Appropriate blocks, wedges and bolus to deliver adequate dose to theplanned target volume was utilized. The site of irradiation, treatmentintent and normal tissue considerations determined the radiation dosageand volume. When stereotactic radiosurgery was used, the dose wasprescribed to the tumor periphery.

Image Analysis. Image acquisition consisted of both planar and singlephoton emission computed tomography (SPECT) studies. Planar studies wereperformed on a dual-head gamma camera (Millenium VG—Variable Geometrymodel available from General Electric Medical Systems of Milwaukee,Wis.) equipped with low energy high-resolution (LEUR) collimators. Thistype of collimator represents a compromise between sensitivity (photoncounting efficiency) and image resolution. Planar nuclear medicineimages were acquired with a 256×256 acquisition matrix (pixel sizeapproximately 0.178 cm/pixel) for 10 minutes. In order to maximizecollimator-gamma camera system sensitivity the source-to-detectorsurface distance was minimized to the extent that patient geometryallows. The spatial distribution of fibrinogen within the planar imagewas measured using region of interest (ROI) analysis. Two different sizeROI's (5×5 pixel, and 15×15 pixel) was used in both the tumor andsurrounding organs and tissues in the patient. The rationale for usingROls with different dimensions is to be able to quantify image countswhile at the same time isolating any possible influence of ROI size onthe results. Tumor-to-background ratios were computed as the ratio ofaverage counts in the tumor region divided by average counts insurrounding organs and tissues, each corrected for background.Background counts was determined based on ROI analysis of a separateplanar acquisition performed in the absence of a radioactive source.

Three-dimensional nuclear medicine SPECT examinations were performedusing the same dual-head gamma camera system. Each SPECT study compriseda 360 scan acquired with a step-and-shoot approach utilizing thefollowing acquisition parameters: three increments between views, a256×256×64 acquisition matrix, LEUR collimation and 60 seconds per view.Images were reconstructed using analytical filtered back-projection andstatistical maximum likelihood techniques with photon attenuationcorrection and post-reconstruction deconvolution filtering forapproximate detector response compensation. In this case, correction forbackground consisted of subtracting counts acquired in a single60-second planar view from all views of the SPECT projection data priorto image reconstruction. SPECT tumor-to-background ratios were computedusing quantitative ROI techniques identical to the planar studies.

Results. Administration of a ^(99m)Tc-labeled biapticide, an RGD peptidemimetic, immediately prior to radiation resulted in tumor binding in 4of 4 patients (Hallahan et al., 2001a). Two patients among this grouphad second neoplasms that were not treated with radiation, and bindingof ^(99m)Tc-labeled biapticide was not observed in the untreated tumor.Administration of the ^(99m)Tc-labeled biapticide within one hourfollowing radiation also failed to show localization of the targetingmolecule to the tumor (Hallahan et al., 2001a).

Example 3 Response of Tumor Blood Vessels to Ionizing Radiation

To determine the response of tumor blood vessels to ionizing radiation,a tumor vascular window and Doppler sonography were used to measure thechange in tumor blood vessels (Donnelly et al., 2001; Geng et al.,2001). Tumors implanted into the window model developed blood vesselswithin 1 week. Tumors were then treated with radiation and the responseof blood vessels was imaged by use of light microscopy. Radiation dosesin the range of 2-3 Gy increased the vascularity within tumors. Incontrast, larger doses of radiation such as 6 Gy reduced tumorvascularity.

Established tumors were studied to determine whether there is adose-dependent change in blood flow following irradiation. Tumors in thehind limb were grown to approximately 1 cm in diameter. Blood flowwithin tumors was measured by use of power Doppler (Donnelly et al.,2001). Tumors were treated with 3 Gy or 6 Gy ionizing radiation, andchanges in tumor blood flow were measured using power Dopplersonography. A radiation dose of 3 Gy achieved an increase in tumor bloodflow. In contrast, radiation doses of 6 Gy or higher markedly reducedtumor blood flow.

Example 4 Preparation of a Recombinant Peptide Library in Phage

A population of DNA fragments encoding recombinant peptide sequences wascloned into the T7 SELECT™ vector (Novagen, Madison, Wis., United Statesof America). Cloning at the EcoR I restriction enzyme recognition siteplaces the recombinant peptide in-frame with the 10B protein such thatthe peptide is displayed on the capsid protein. The resulting readingframe requires an AAT initial codon followed by a TCX codon.

The molar ratio between insert and vector was 1:1. Size-fractionatedcDNA inserts were prepared by gel filtration on sepharose 4B and rangedfrom 27 base pairs to 33 base pairs. cDNAs were ligated by use of theDNA ligation kit (Novagen, Madison, Wis., United States of America).Recombinant T7 DNA was packaged according to the manufacturer'sinstructions and amplified prior to bioscreening in animal tumor models.The diversity of the library was 10⁷.

Example 5 In Vivo Screening for Peptide Ligands to Radiation-InducibleMolecules

GL261 murine glioma cells and Lewis lung carcinoma cells were implantedinto the hind limb of C57BL6 mice (Hallahan et al., 1995b; Hallahan etal., 1998; Hallahan & Virudachalam, 1999).

To determine the optimal time at which peptides bind within tumors,phage were administered at 1 hour before, at 1 hour after, and at 4hours after irradiation of both LLC and GL261 tumors. Phage wererecovered from tumors when administered 4 hours after irradiation. Phageadministered 1 hour before or 1 hour after irradiation were notrecovered from tumors. These data indicate that the optimal time ofadministration is beyond 1 hour after irradiation.

For in vivo screening, tumors were irradiated with 3 Gy andapproximately 10¹⁰ phage (prepared as described in Example 4) wereadministered by tail vein injection into each of the tumor bearing miceat 4 hours following irradiation. Tumors were recovered at one hourfollowing injection and amplified in BL21 bacteria. Amplified phage werepooled and re-administered to a tumor-bearing mouse following tumorirradiation. The phage pool was sequentially administered to a total of6 animals. As a control, wild type phage lacking synthetic peptideinserts were identically administered to a second experimental group ofanimals.

To determine the titer of phage binding in a tumor or in normal tissue,recovered phage were amplified in BL21 bacteria. Bacteria were platedand the number of plaques present were counted. To determine the totalphage output per organ, the number of plaque forming units (PFU) on eachplate was divided by the volume of phage plated and the weight of eachorgan. Normal variation was observed as a 2-fold difference in PFU.

In the present study, background binding within tumor blood vessels wasapproximately 10⁴ phage. Phage that bound to the vasculature withinirradiated tumors show enrichment in the tumor relative to other organsand enrichment in the irradiated tumor relative to the control phagewithout DNA insert. Phage that home to irradiated tumors showed abackground level of binding in control organs that was lower thancontrol phage without DNA insert.

Following 6 rounds of in vivo screening, fifty recombinant phagepeptides that bound within irradiated tumors were randomly selected forfurther analysis. The nucleic acid sequence encoding recombinant phagewas amplified by PCR using primers set forth as SEQ ID NOs: 14-15(available from Novagen, Madison, Wis., United States of America). Anindividual phage suspension was used as template. Amplified peptideswere sequenced using an ABI PRISM 377 sequencer (PE Biosystems, FosterCity, Calif., United States of America). The sequences of the encodedpeptides are listed in Table 1. Several conserved subsequences werededuced from the recovered peptides and are presented in Table 2.

Peptide sequences recovered from both tumor types include NHVGGSSV (SEQID NO: 1), NSLRGDGSSV (SEQ ID NO: 2), and NSVGSRV (SEQ ID NO: 4). Of thepeptide sequences recovered from 6 irradiated tumors, 56% had thesubsequence GSSV (SEQ ID NO: 5), 18% had the sequence RGDGSSV (SEQ IDNO: 6), and 4% had the sequence GSRV (SEQ ID NO: 7). Approximately 22-40of 10⁶ injected phage were recovered from irradiated tumors having apeptide insert comprising the subsequence GSSV (SEQ ID NO: 5). Bycontrast, no phage were from irradiated tumors following administrationof 10⁶ wild type phage.

TABLE 1 Peptides Identified by In Vivo Screening of LLC and GL261 Tumors Number  of Phage Recovered from Number of PhageGL261  Recovered from LLC tumors Peptide Sequence tumors (Frequency)(Frequency) NHVGGSSV 7 (28%) 12 (48%) (SEQ ID NO: 1) NSLRGDGSSV 7 (28%)2 (8%) (SEQ ID NO: 2) NSVRGSGSGV 7 (28%) 0 (SEQ ID NO: 3) NSVGSRV1 (4%)  3 (12%) (SEQ ID NO: 4) Unique Sequences 3 (12%) 8 (32%)

TABLE 2 Conserved Motifs within Peptides Identified by In Vivo ScreeningConserved Sequence Frequency of Recovery GSSV (SEQ ID NO: 13) 56%GSXV (SEQ ID NO: 8) 78% NSXRGXGS (SEQ ID NO: 9) 32% NSV (SEQ ID NO: 10)22% NSXR (SEQ ID NO: 11) 32% NXVG (SEQ ID NO: 12) 46%

Example 6 Peptide Targeting in Additional Tumors

The binding properties of phage encoding NHVGGSSV (SEQ ID NO: 1),NSLRGDGSSV (SEQ ID NO: 2), NSVRGSGSGV (SEQ ID NO: 3), and NSVGSRV (SEQID NO: 4) were additionally characterized in a B16F0 melanoma model.Peptides set forth as SEQ ID NOs: 1 and 2 bound within the melanoma,lung carcinoma, and glioma tumor models. SEQ ID NO: 3 bound withinglioma and melanoma, and SEQ ID NO: 4 bound within lung carcinoma andglioma.

Example 7 Characterization of Peptide Binding to Irradiated Tumors

To determine where recombinant peptides bind in tumor blood vessels, thebiodistribution of biotinylated peptides was assessed. Tumors weretreated with 3 Gy and biotinylated peptides were administered by tailvein at 4 hours following irradiation. Tumors were recovered 30 minutesfollowing administration of biotinylated peptides. Tumors were snapfrozen and sectioned on a cryostat. Frozen sections were then incubatedwith Avidin-FITC (fluorescein isothiocyante) and imaged by fluorescentmicroscopy. Recombinant peptides (for example, those set forth inTable 1) were observed to bind the vascular endothelium within tumorblood vessels.

The anti-α_(2b)β₃ monoclonal antibody was administered by tail vein todetermine whether this receptor is required for recombinant phagebinding in irradiated tumors. Phage encoding SLRGDGSSV (SEQ ID NO: 5) onthe capsid protein were injected immediately after blocking antibody orcontrol antibody. Phage were recovered from the tumor and controlsorgans and quantified by plaque formation. Radiation induced a 4-foldincrease in phage binding in tumor. Blocking antibody eliminatedinduction of phage binding, while control antibody to P-selectin (onactivated platelets) did not reduce phage binding. Thus, the tumorbinding activity of targeting peptide SLRGDGSSV (SEQ ID NO: 5) isdependent on its interaction with the α_(2b)β₃ receptor.

Example 8 Production of a Phage-Displayed scFv Antibody Library

A phage-displayed antibody library was constructed based upon previouslypublished methodologies (see Pope et al., 1996). Briefly, spleens fromoutbred newborn and three-to-four week old mice and rats were used as asource of antibody-encoding genetic material to produce a library ofabout 2×10⁹ members. The antibody-encoding genetic material was clonedinto the pCANTAB phagemid vector.

The pCANTAB vector contains an amber stop codon that is locateddownstream of the scFv coding sequences and upstream of the M13 gene IIIcoding sequences. E. coli TG1 cells (a sup E strain of E. coli) containa suppressor tRNA that inserts a glutamic acid residue in response to anUAG (amber) stop codon. The amber stop codon is about 14% efficient.Therefore, the scFv antibody amino acid sequences will be fused to M13phage gene III amino acid sequences about 14% of the time, and will beproduced as a soluble, non-fusion protein about 86% of the time when thelibrary is grown in TG1 cells. In contrast, E. coli strain HB2151 doesnot contain the amber stop codon, and thus only soluble non-fused scFvwill be produced when the library is grown in HB2151.

Example 9 In Vivo Screening for Antibody Ligands to Radiation-InducibleNeoantigens

A phage library comprising diverse single chain antibodies was preparedin M13 phage. Briefly, nucleotide sequences encoding antibody V_(L) andV_(H) regions separated by a (Gly₄Ser)₃ linker were fused to M13 geneIII nucleotide sequences. When a recombinant M13 carrying theseantibody-gene III fusions infects an appropriate host bacterium, thehost produces recombinant M13 phage that display scFv antibodypolypeptides (V_(L)-linker-V_(H)) fused to gene III protein.

The phage library was exposed to the radiation-inducible neoantigensP-selectin (also called CD62P; GENBANK™ Accession No. P98109) and/orplatelet membrane glycoprotein IIB (also called CD41; GENBANK™ AccessionNo. P08514) immobilized on glass slides. Phage were selected based onantigen binding, and selected phage were pooled as a biased library. Forrepresentative in vitro screening methods, see Fowlkes et al., 1992;Haaparanta & Huse, 1995; Jung & Pluckthun, 1997; Peter et al., 2000;Holzem et al., 2001; Chiu et al., 2000.

Phage identified by in vitro screening were tested on Westernimmunoblots to confirm binding to the P-selection and platelet membraneglycoprotein IIB neoantigens. Phage that specifically bound P-selectinand platelet membrane glycoprotein IIB were subsequently used for invivo screening of irradiated tumors as described in Example 5. Wild typephage were used as internal controls. Antibodies having substantialaffinity for irradiated tumors were identified by observing an increasednumber of phage in the irradiated tumor when compared to a number ofphage in a control organ (e.g., liver and lung). Phage antibodies withthe greatest affinity for tumors were identified using the formula:number of phage in irradiated tumor/number of phage in each organ.

Several antibodies that bound P-selectin and several other antibodiesthat bound platelet membrane glycoprotein IIB were recovered followingin vivo screening to irradiated tumors. Representative targetingantibodies identified by this method include the single chain antibodiesset forth as SEQ ID NOs: 18, 20, 22, and 24 (encoded by SEQ ID NOs: 17,19, 21, and 23 respectively), that recognize the radiation-inducibleneoantigens P-selectin and platelet membrane glycoprotein IIB.

Examples 10-13 Delivery Vehicles for Use in X-Ray Guided Drug Delivery

Examples 10-13 pertain to site-specific drug delivery systems that bindto irradiated tumor blood vessels. In Examples 10-13,radiation-inducible targets, including integrin β₃ (component ofreceptors GPIIb/IIIa and α_(v)β₃), for the delivery vehicles aredescribed. The drug delivery methods and compositions of the presentlydisclosed subject matter, including those described in Examples 10-13,are applicable to all vascularized neoplasms, thereby eliminating theproblem of tumor-type specificity.

As disclosed herein above, ionizing radiation can be used to guide drugsto specific sites such as neoplasms or aberrant blood vessels. Whenblood vessels are treated with ionizing radiation, they respond byexpressing a number of cell adhesion molecules and receptors thatparticipate in homeostasis (referred to herein as “radiation-inducibletargets”). Mass spectrometry has been used to study protein expressionwithin tumor blood vessels. A number of proteins have beencharacterized. One such protein is the integrin β₃. Other examples ofradiation-inducible molecules in blood vessels include ICAM-1, Endoglin,E-selectin, and P-selectin.

Antibody binding to radiation-inducible targets such as the celladhesion molecules (CAMs) ICAM-1, E-selectin, and P-selectin, Endoglin,and the β₃ integrin subunit is also disclosed herein. Ionizing radiationinduces oxidative injury in the endothelium. The endothelium responds tomaintain homeostasis by preserving the barrier function in bloodvessels. This is accomplished by activation of inflammation and plateletaggregation. The mechanism by which radiation activates thesehomeostatic responses is, in part, through the induction of celladhesion molecules. This requires the activation of the transcriptionfactor NFκB, which regulates transcription of the ICAM-1 and E-selectingenes (Hallahan, 1995a; Hallahan, 1996b; Hallahan, 1998b). ICAM-1,E-selectin, and P-selectin are induced by X-irradiation of theendothelium and bind to receptors on circulating leukocytes to initiateinflammation.

P-selectin (GMP140, CD62P) contributes to the inflammatory responsefollowing translocation from the cytoplasm of the vascular endotheliumto the luminal surface of irradiated blood vessels. P-selectin is a celladhesion molecule (CAM) that is sequestered in storage reservoirs withinthe vascular endothelium and granules in platelets. This CAM istranslocated to the blood-tissue interface of the endothelium, and isnot released from storage reservoirs, but remains tethered to theendothelial cell membrane (Johnston, 1989). P-selectin is rapidlytranslocated to the vascular lumen after tissue injury to initiate theadhesion and activation of platelets and leukocytes (Malik, 1996).

As disclosed herein, the histologic pattern of P-selectin expression inirradiated tumor blood vessels was studied and it was observed thatP-selectin was localized within the endothelium of tumor vessels priorto treatment. At one hour following irradiation, P-selectin waslocalized to the lumen of blood vessels. P-selectin localization to thevascular lumen was present in all tumors and all species studied.Irradiated intracranial gliomas showed P-selectin localization to thevascular lumen within one hour, whereas blood vessels in normal brainshowed no P-selectin staining in the endothelium and no localization tothe irradiated vascular lumen.

An additional paradigm of radiation-inducible targets for drug deliveryis activation (conformational changes) of receptors within irradiatedblood vessels. The integrin β₃ (a component of receptor GPIIID/IIIa,α_(2b)β₃) is activated and accumulates in the lumen of irradiated tumorblood vessels. Glycoproteins (GP) IIb and IIIa are members of theintegrin superfamily and are the predominant surface glycoproteins inthe platelet plasma membrane (Hawiger & Timmons, 1992). Theseglycoproteins form a heterodimer GPIIID/IIIa (Carrell, 1985). Plateletscontain several integrins, including the collagen receptor α₂β₁, thefibronectin receptor α₅β₁, and the vitronectin receptor α_(v)β₃. Ofthese integrins, GPIIb/IIIa appears to be unique in that it is the onlyintegrin that is restricted to platelets and cells of megakaryoblasticpotential.

Human fibrinogen interacts with binding sites exposed on GPIIID/IIIa ofstimulated platelets through the tentacles present on Y and a chains(Hawiger, 1982). The 12-residue carboxyl-terminal segment of the Ychain, encompassing residues 400-411, was pinpointed by Hawiger andothers as the platelet receptor recognition domain. See e.g. Hawiger &Timmons, 1992. Hawiger also showed that the sequence—RGD (amino acids95-98) and (amino acids 572-575) are involved in the interaction ofhuman fibrinogen a chain with receptors on activated platelets (Hawigeret al., 1989), but these regions are not essential for fibrinogenbinding. Both domains contain the sequence RGD, identified previously asthe cell recognition site of fibronectin. The presence of three domainson each half of the fibrinogen molecule, provides conditions for tighterbinding of fibrinogen to platelets and for their subsequent aggregation

A fibrinogen molecule comprises three pairs of nonidentical chainsarranged in an anti-parallel configuration. The platelet receptorrecognition domains encompass sequence 400-411 on the chain. RGDsequences 95-98 and 572-575 on the chain bind, but are not essential.One fibrinogen molecule can be engaged in trans and cis interactionswith platelet receptor GPI Ibillla.

Thus, identified herein are several radiation-inducible target proteinsin blood vessels. These include E-selectin, ICAM-1, P-selectin, and theβ₃ integrin, which are expressed at radiation doses as low as 2 Gy.E-selectin and ICAM-1 are induced at the level of transcription in thevascular endothelium in response to ionizing radiation exposure. Levelsof E-selectin protein and RNA induction following irradiation ofvascular endothelial cells increase seven- to ten-fold. Likewise, levelsof ICAM protein and RNA induction following irradiation of theendothelium increase approximately three-fold.

In addition to the transcriptional induction of genes in the vascularendothelium, preexisting proteins are translocated or activatedfollowing X-irradiation. For example, as disclosed herein, P-selectin isstored in a storage reservoir (Weibel Palade bodies), which undergoexocytosis in response to X-irradiation. P-selectin expression on thesurface of endothelial cells in response to ionizing radiation has beenobserved. P-selectin accumulation within the lumen of tumormicrovasculature following tumor irradiation was also observed. Thisresponse occurs at therapeutic doses of radiation (2 Gy) and typicallyoccurs within one hour of X-irradiation.

β₃ also accumulates within the lumen of blood vessels in response toradiation. β₃ is associated with integrins α_(v) or α_(2b) to formheterodimers α_(2b)β₃ and α_(v)β₃. The heterodimer α_(2b)β₃ is thecomponent of a receptor on activated platelets, glycoprotein IIb/IIIa(GPIIID/IIIa), while α_(v)β₃ is the vitronectin receptor. As disclosedherein, while several other radiation-inducible molecules can betargeted within tumor blood vessels, the β₃ target for drug delivery isan exemplary target for site-specific peptide binding within tumor bloodvessels following irradiation.

As set forth in Examples 10-13, peptides and antibodies that bind to β₃have been studied. β₃-binding proteins have been conjugated tofluorochromes and radionuclides to determine whether specific binding ofpeptides occurs within irradiated tumors. Immunofluorescent andimmunohistochemical staining of β₃ within the lumen of blood vesselsimmediately following irradiation has been observed. Drug delivery toirradiated tumors in accordance with the presently disclosed subjectmatter has been studied through the analysis of ligands to β₃(vitronectin, von Willebrand factor, fibronectin, and fibrinogen). ¹³¹Iwas conjugated to these ligands to determine the biodistribution intumor bearing mice. These studies demonstrated that ¹³¹I-fibrinogenbinds specifically to tumors following exposure to ionizing radiation.

Immunoconjugates directed to radiation-inducible neoantigens inaccordance with the presently disclosed subject matter circumvent thelimitation associated with attempts in the prior art to prepareimmunoconjugate delivery vehicles in that prior art immunoconjugates arelimited to certain tumor types. In contrast, because antigens that areinduced in irradiated vessels in all tumor types have been selected foruse in the methods and compositions of the presently disclosed subjectmatter, all tumor types can be targeted. This is possible because it hasbeen observed in that the endothelium and blood components respond tooxidative stress in a similar, if not identical manner in all tumors.

Examples 10-13 provide data that demonstrates improved bioavailabilityand biodistribution of therapeutic agents to irradiated tissues inanimal models. The methods and compositions of the presently disclosedsubject matter thus provide for an increase in the bioavailability oftherapeutic agents at biologically active sites, and for a reduction intoxicity by directing treatment specifically to the neoplasm or the siteof angiogenesis. Thus, an aspect of the presently disclosed subjectmatter is to target drug delivery to these radiation-inducible moleculesthrough antibody conjugate delivery vehicles, protein conjugate deliveryvehicles and peptide conjugate delivery vehicles.

Site-specific drug delivery to radiation-inducible antigens is adaptableto many compounds and therapeutic approaches. In this regard, anysuitable therapeutic agents, including but not limited to cytotoxins,biologicals, gene therapy vectors, and radionuclides, can beincorporated into a delivery vehicle of the presently disclosed subjectmatter.

Materials and Methods Employed in Examples 10-13

Linking Compounds. Linking compounds include1,3,4,6-tetrachloro-3a,6a-diphenylglcouril (a reagent sold under theregistered trademark IODO-GENC), and MPBA, each available from PierceBiotechnology, Inc. (Rockford, Ill., United States of America). TheIODO-GEN® reagent reacts with tyrosine residues, while MPBA reacts withcysteine residues, both of which are not on the peptide HHLGGAKQAGDV(SEQ ID NO: 16). An advantage of the IODO-GEN® reagent is that it issupplied in coated tubes and beads to eliminate contamination of theinjectable material, whereas MPBA is in powder form. Initial experimentsuse the IODO-GEN® reagent to iodinate a poly-tyrosine peptide derivativeof HHLGGAKQAGDV-SGSGS (SEQ ID NO: 26), HHLGGAKQAGDV-SGSGS-YYYYY (SEQ IDNO: 28), and additional experiments use MPBA to iodinate poly-Cys.

Preparation and Radioiodination of Peptides. An IODO-GEN®-platedreaction vessel (Pierce Biotechnology, Inc.) is rinsed with a smallamount of sterile saline to remove any loose microscopic flakes of theiodination reagent. The desired amount of carrier-free ¹²⁵I sodiumiodide, a specific activity of 100 mCi/mg protein, is added to thereaction vessel, followed by the reconstituted peptides suspension. Thereaction vessel is then sealed off and the reaction is allowed toproceed for 20 minutes at room temperature with constant gentleagitation of the reaction vessel. The iodination process is terminatedby removing the reaction mixture from the reaction vessel into acentrifugation tube. The reaction mixture is centrifuged at 3,000 rpmfor 15 minutes. The supernatant is removed and the residue isreconstituted in 5 ml sterile normal saline.

Pinhole Gamma Camera Imaging of Peptide Biodistribution. A dedicatedresearch single-head gamma camera (20 cm×40 cm active imaging area)fitted with a cone-shaped pinhole collimator is used for nuclearmedicine animal imaging experiments. The pinhole collimator, equippedwith a 4 mm aperture Tungsten insert, is used to acquire pre-treatmentand serial, post-treatment follow-up images of each animal in order todetermine the temporal distribution of peptide in vivo. Each pinholeacquisition comprises a planar view acquired for 3 minutes using a256×256 pixel acquisition matrix. In order to maximize pinholecollimator-gamma camera system sensitivity, a source-to-aperturedistance on the order of 2 cm to 5 cm is maintained. The spatialdistribution of peptide within each image is measured usingquantitative, region of interest (ROI) analysis. Two different size ROlsare used in both the tumor region and mouse background in order toquantify image counts and isolate any possible influence of ROI size onquantification. A 2×2 (small) and 11×11 (large) pixel ROI are used torecord image counts in the tumor and other organs in the mouse. Theangular dependence of pinhole efficiency is measured using a flat,uniform sheet source of activity. Image counts are then corrected fordecay and this geometric effect.

Statistical Considerations. Internal controls are established in eachanimal by use of an untreated control tumor implanted on the left hindlimb and irradiation of the right hind limb tumor, as described inHallahan, 1995b and Hallahan, 1998.

Sample Size and Power Analysis. In order to calculate the statisticalsignificance of differences between groups of mice, eight mice arestudied at each time to determine statistical significance. In general,a sample size of eight per group gives about 80% of power to detect adifference of 1.5-fold standard deviations in the interesting parametersbetween two groups with a two-sided statistic equal to 5%.

Statistical Analysis Plan. Pharmacokinetic parameters are presented intabular and graphic form. Pharmacokinetic parameters such as maximalplasma concentration, time of maximal concentration, and area under theplasma concentration time curve are determined using non-compartmentalmethods. Statistical analyses are performed using the General LinearModel method of the Statistical Analysis System (SAS). If significantdifferences are indicated by the ANOVA analysis, the Waller-DuncanK-ratio t-test procedure is used for pairwise comparisons of meanpharmacokinetic parameter values.

For the single time point data, tests of hypotheses concerningcorrelation between imaging results and results are completed using thepaired t-test or Wilcoxon Signed-Rank test for the interestingcontinuous parameters or the McNemar's Chi-square test for theinteresting categorical parameters. For either count or binary multipletime points data, tests concerning correlation between imaging resultsand pharmacokinetic results are made using the Generalized EstimatingEquation (GEE) method statistical procedure for longitudinal dataanalysis with multiple observable vectors for the same subject (Diggle,1994; Liang, 1986). For continuous multiple time points data, testsconcerning correlation between groups are completed using therestricted/residual maximum likelihood (REML)-based repeated measuremodel (mixed model analysis; Jennrich, 1986) with various covariancestructure.

The statistical analyses are completed by SAS 6.12 statistical program,or SAS IML macro in this project. Computer connections, when necessary,are attained via a Novell network using the Internet Packet exchange(IPX) protocol.

Example 10 X-Ray-Guided Drug Delivery Via Antibody Delivery Vehicles

Following platelet activation, several antigens are expressed on thesurface of platelets. Indeed, it has been observed that irradiation ofanimal tumors increases the expression of platelet antigens such asP-selectin and GPIIb/IIIa. As disclosed herein above, antibodies can beconjugated to radionuclides, cytotoxic agents, gene therapy vectors,liposomes, and other active agents. In this Example, the administrationof radioimmunoconjugate delivery vehicles against platelet antigensfollowing irradiation of tumors is disclosed.

Anti-GPIIb/IIIa antibodies (R&D Systems, Inc., Minneapolis, Minn.,United States of America) are labeled with ¹³¹I using IODO-GEN® reagent(Pierce Biotechnology, Inc., Rockford, Ill., United States of America).Labeled antibody is separated from free iodine by use of columnchromatography. Radioimmunoconjugates are injected into mice by tailvein. Hind limb tumors are implanted and treated as described hereinabove. The optimal time of administration of radioimmunoconjugates isdetermined.

In separate experiments, procoagulants such as DDAVP are alsoadministered to enhance radioimmunoconjugate binding to activatedplatelets in irradiated tumors. Mouse subjects are imaged by gammacamera as described herein above. PHOSPHORIMAGER™ plates and histologicsections with immunohistochemistry as described herein above are used tovalidate image processing. In the event that certainradioimmunoconjugates do not achieve specific activity within tumorsthat is sufficient to image or treat tumors, multiple radionuclides areincorporated into the antibody delivery vehicles.

In additional experiments, Fab′ fragments of anti-GPIIIa and anti-GPIIbantibodies are also employed in binding in a site-specific manner toirradiated tumors. It is shown herein that anti-GPIIIa antibody stainingin blood vessels following X-irradiation. There are two approaches inproducing antibodies for site-specific binding. The first is cleavage ofthe IgG antibody to form the Fab′ fragment. The second approach is theuse of phage antibodies to GPIIIa and GPIIb that are produced in theVanderbilt Cancer Center Molecular Discovery Core Laboratory usingphage-display techniques. Each of these approaches yields low molecularweight antibodies that can be efficiently produced for clinical studies.Specificities of the GPIIIa (integrin β₃) antibodies and antibodyfragments are compared to the specificities of the GPIIb antibodies andantibody fragments to establish potentially useful reagents and in thatGPIIIa is also found in α_(v)β₃.

Experimental Design. The anti-GPIIIa and anti-GPIIb antibodies (R&DSystems, Inc.) are cleaved to form the Fab′ fragment. This fragment isisolated from the Fc fragment by columns. In addition, GPIIIa protein isscreened with a phage library within the Vanderbilt Cancer CenterMolecular Discovery Core Laboratory. Antibody from phage is grown up inthe bacteria. Antibodies are then studied for binding in irradiatedtumors. Antibodies are labeled with ¹³¹I using IODO-GEN® reagent asdescribed above. The molar ratio of ¹³¹I to antibody is optimized toavoid potential reduction in the affinity of antibody binding due to¹³¹I.

Tumors are implanted and irradiated as described herein.Radio-immunoconjugates are administered immediately after irradiationusing tail vein injection. Eight mice are randomly assigned intoexperimental and control groups. Imaging and quantification of ¹³¹I areperformed as described above. Statistical analysis is performed asdescribed above.

Positive control groups. Radiolabeled fibrinogen is administered toirradiated tumor bearing mice and compared to radioimmunoconjugates.These mice are randomly assigned into groups during the same experimentas radioimmunoconjugates.

Negative control groups. Non-irradiated control tumors are implanted inthe left hind limb of all mice. Secondly, radiolabeled anti-_(v) andanti-human IgG antibodies are administered to tumor bearing micefollowing irradiation to verify that antibody binding to irradiatedtumors is not a generalized phenomenon.

Example 11 X-Ray-Guided Drug Delivery Targeted to Radiation-Inducible

Neoantigens in Blood Vessels

Radiation-inducible targets for drug delivery systems will be mostuseful if they are not tumor-specific. The vascular endothelium is anessential component to nearly all neoplasms. As disclosed herein above,radiation response is similar across a wide range of tumor types. Inparticular, P-selectin exocytosis, von Willebrand Factor release, andplatelet aggregation are observed within all tumor blood vesselsfollowing irradiation. In this Example, antibody delivery vehicles forX-ray-guided drug delivery to the vascular endothelium of tumors aredisclosed. Antibody delivery vehicles adhere to antigens released intothe lumen and are thus obstructed from circulating beyond the confinesof the tumor. In view of the targeting of vascular endothelium, thisExample is also illustrative of the methods of treating angiogenesis inaccordance with the presently disclosed subject matter disclosed hereinabove.

Additionally, one level of radiation-inducible expression of receptorsand adhesion molecules is the activation of inactive receptors followingirradiation of tumor blood vessels. Tumors in the hind limb of mice weretreated with 2 Gy ionizing radiation followed by sectioning andimmunohistochemical staining for the.₃ integrin in the tumor sections.The observed histologic pattern of staining showed that both plateletsand endothelium stain with anti-133 antibody after irradiation, but notprior to irradiation. Thus, therapeutic doses of irradiation (2 Gy) wereand are sufficient to induce the accumulation of integrin.₃ within tumorblood vessels within 1-4 hours of irradiation.

Hind limb tumors are implanted into mice and treated with radiation asdescribed in Hallahan et al., 1998a. Radioimmunoconjugate deliveryvehicles are prepared using anti-E-selectin and anti-P-selectinantibodies (R&D Systems, Inc.), IODO-GEN® reagent (Pierce Biotechnology,Inc.) and ¹³¹I. Radiolabeled antibodies are separated from free ¹³¹I byuse of column chromatography. The delivery vehicles are injected viatail vein into mice with hind limb tumors following treatment withirradiation. Mice are imaged with gamma camera imaging as describedherein above. Image processing is validated by use of PHOSPHORIMAGER™plates, immunofluorescence, and immunohistochemistry as described hereinabove.

One potential limitation of this embodiment of the presently disclosedsubject matter is that anti-E-selectin antibody binding occurs inuntreated normal tissues such as the lung. The importance of validationof the tumor specificity for radioimmunoconjugate delivery vehicles isthat the ideal radiation-inducible antigens have substantially noconstitutive expression in any tissue, but prolonged expression in tumorblood vessels. Thus, pharmacokinetics and biodistribution of theanti-E-selectin and anti-P-selectin antibody delivery vehicles are alsodetermined.

Example 12 X-Ray-Guided Drug Delivery by Use of a Twelve Amino AcidSegment of the γ Subunit of Fibrinogen

This Example pertains to the use of the dodecapeptide HHLGGAKQAGDV (SEQID NO: 16), a segment of they subunit of fibrinogen, to achievesite-specific binding to irradiated tumors. This peptide segment of thecarboxyl terminus of the fibrinogen γ chain binds to GPIIID/IIIafollowing platelet activation. The fibrinogen binding sequence(HHLGGAKQAGDV; SEQ ID NO: 16) is sufficient for site-specificlocalization to irradiated tumors.

Observations. The peptide sequence within fibrinogen that binds to theactivated GPIIID/IIIa receptor is the dodecapeptide HHLGGAKQAGDV (SEQ IDNO: 16). To determine whether HHLGGAKQAGDV (SEQ ID NO: 16) binds inirradiated tumors, applicant utilized the peptide HHLGGAKQAGDV (SEQ IDNO: 16) linked to biotin by a serine-glycine linker(HHLGGAKQAGDV-SGSGSK-biotin; SEQ ID NO: 30). This peptide wassynthesized in the Vanderbilt University Peptide Core Lab andbiotinylated at the carboxyl terminus. The resultingHHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) was administered by tail veininjection into tumor bearing mice. B16F0 tumors in the hind limb weretreated with sham irradiation (control), 4 Gy irradiation followed byHHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) injection, orHHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) followed by tumor irradiation(4 Gy). Tumors were frozen at 4 hours and sectioned for fluorescencestaining. Avidin-FITC was incubated with tumor sections and imaged by UVmicroscopy. Avidin-FITC stained blood vessels were observed inirradiated tumors, but not in untreated control. Moreover, it was foundthat HHLGGAKQAGDV (SEQ ID NO: 16) administration prior to irradiation isa more efficient schedule of administration as compared to radiationbefore dodecapeptide administration.

Design of Iodination Experiments. Tumors are implanted and irradiated asdescribed herein above. The synthetic dodecapeptide encompassing thesequence HHLGGAKQAGDV (SEQ ID NO: 16) on the carboxyl-terminal segmentof fibrinogen γ chain binds to GPIIID/IIIa is prepared, and a peptidetail for radioiodination (SGSGS-YYYYY; SEQ ID NO: 32) is added. Thepeptide tail is commercially available from PeptidoGenic Research & Co.(Livermore, Calif., United States of America). A sample from each batchis sequenced in accordance with standard techniques for quality control.

HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) is labeled with ¹³¹I usingIODO-GEN® reagent as described above. When tumors are grown to 0.5 cm indiameter, the tail vein of each mouse subject is cannulated and¹³¹I-labeled HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) is injected. Theinjection tubing and syringe is counted after the injection to measureresidual ¹³¹I. Immediately after administration of ¹³¹I-peptide, tumorsare irradiated using techniques described herein and by Hallahan et al.,1998. Mice are imaged by gamma camera imaging at 1 and 24 hours afterirradiation. ¹³¹I-labeled HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28)binding to tumors is quantified by gamma camera imaging and direct wellcounts from excised tumors as described above. Tissue sections of allorgans are analyzed. Eight tumor-bearing mice are randomly assigned intoeach of the experimental and control groups. Statistical considerationsare addressed as described above.

Positive control groups. Radioiodinated-fibrinogen is administered toirradiated tumor bearing mice and compared to radioiodinated-peptide.These mice are randomly assigned into groups during the same experimentas radiolabeled peptides.

Negative control groups. Non-irradiated control tumors are implanted inthe left hind limb of all mice. Secondly, radiolabeledSGSGSGSSGSGSSGSGS-YYYYY (SEQ ID NO: 33) are administered to tumorbearing mice following irradiation to verify that peptide binding toirradiated tumors is not a generalized phenomenon.

It is noted that the three-dimensional conformation of fibrinogen mightfacilitate site-specific binding to irradiated tumors. Alternatively,¹³¹I labeling might interfere with peptide binding to GPIIb/IIIa. Alonger peptide linker and fewer Tyr residues are options that areemployed in each case.

Example 13 Liposome Delivery Vehicle Comprising Twelve Amino AcidSegment of the γ Subunit of Fibrinogen

This Example pertains to the preparation of liposomes that areconjugated to the dodecapeptide HHLGGAKQAGDV (SEQ ID NO: 16), a segmentof the γ subunit of fibrinogen, to achieve site-specific binding toirradiated tumors.

In initial experiments,1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine perchlorate(Dil), a lipid fluorescent marker, was added to liposome-fibrinogenconjugates and injected by tail vein. As a control, liposomes withoutfibrinogen conjugation were injected. These produced no increase influorescence in irradiated tumors. Fluorescence within blood vessels oftumors treated with ionizing radiation was observed for theliposome-fibrinogen conjugates. These findings support site-directeddrug delivery to the fibrinogen receptor in irradiated tumors.

Cationic liposomes can be conjugated to antibodies and peptides(Kirpotin et al., 1997); however, these liposomes bind to lipophilicproteins in the serum, which reduces the circulation time. Therefore,polyethylene glycol (PEG) is used to coat the drug delivery systems. PEGprolongs circulation time (Nam et al., 1999; Koning et al., 1999).

In this Example, HHLGGAKQAGDV (SEQ ID NO: 16) is conjugated to liposomesand encapsulated by PEG. It is then determined whether both large MWtherapeutic proteins and small MW cytotoxic compounds can be localizedto irradiated tumors by liposomes conjugated to HHLGGAKQAGDV (SEQ ID NO:16). The linking peptide SGSGS (SEQ ID NO: 31) is placed at theC-terminus, which is linked to liposomes. Liposomes are conjugated tothe SH on Cys at the C-terminus. The biodistribution ofHHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-liposome is studied and the lengthof the linking peptide is altered as necessary. In the event that PEGwill not achieve membrane fusion that is comparable to cationicliposomes, the length of the linking peptide is also altered asnecessary.

Preparation of HHLGGAKQAGDV (SEQ ID NO: 16)-Long Circulatory Liposomes.Two methods of conjugating liposomes to peptides are employed. The firstmethod conjugates the liposome to the N-terminus, and thus the linkingpeptide is placed at the N-terminus. This method arranges the conjugatein the following configuration: liposome-SGSGS-HHLGGAKQAGDVC (SEQ ID NO:27). The second method conjugates the liposome to the C-terminus of thepeptide. This method is facilitated by placing a Cys residue at theC-terminus. This method arranges the conjugate into the configuration:HHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-liposome. These two methods providealternatives in the event that one configuration is useful forsite-specific drug delivery over the other configuration. These methodsare also applicable to larger polypeptides and proteins, includingfibrinogen itself.

Method 1

Step (1) Synthesis of maleimide-PGE-PE

The lipophilic SH reactive reagent with a long spacing arm issynthesized from maleimide-PEG 2000-NHS ester (Prochem, High Point,N.C., United States of America), dioleoylphosphatidylethanolanime (DOPE,Avanti Polar Lipids, inc., Alabaster, Ala., United States of America),and triethylamine in chloroform (1:1:1.5). Resulting maleimide-PEG2000-DOPE is purified by flash column.

Step (2) Preparation of thiolated HHLGGAKQAGDV (SEQ ID NO: 16)

Under stirring, to a solution of HHLGGAKQAGDV (SEQ ID NO: 16; 2 mg/mL)in 0.01 M HEPES 0.15 M NaCl buffer pH 7.9, containing 10 mM EDTA and0.08% sodium azide, is added in five-fold excess of freshly preparedTraut's Reagent in the same buffer. Reaction is performed for 30 minutesat 0° C. Thiolated HHLGGAKQAGDV (SEQ ID NO: 16) is then purified using adesalting PD-10 column (Amersham Biosciences).

Preparation of maleimide-containing long circulating liposomes withfluorescent labels. PGE 2000-PE, cholesterol, Dipalmitoyl phosphocholine(Avanti Polar Lipids), Dil, and maleimide-PEG-2000-DOPE is dissolved inchloroform and mixed at a ratio of 10:43:43:2:2 in a round bottom flaskas described in Leserman, 1980. The organic solvent is removed byevaporation followed by desiccation under vacuum for 2 hours. Liposomesare prepared by hydrating the dried lipid film in PBS at a lipidconcentration of 10 mM. The suspension is then sonicated 3×5 minutesuntil clear, forming unilamellar liposomes of 100 nM in diameter.

Conjugation of thiolated HHLGGAKQAGDV (SEQ ID NO: 16) to maleimidecontaining liposomes. Prepared vesicles and thiolated protein is mixedin 10 mm HEPES, 0.15 M NaCl, and EDTA pH 6.5. The final concentrationsfor proteins and liposomes are 0.25 g/L and 2.5 mM, respectively. Themixture is incubated for 18 hours at room temperature and vesicles areseparated from unconjugated protein by gel filtration on a SEPHAROSE®4B-CL column (Amersham Biosciences).

Method 2

To conjugate the peptide to long-circulating liposomes, a peptide with aCys residue on the C-terminal is synthesized (PeptidoGenic Research &Co., Livermore, Calif., United States of America). A bifunctional PEG(molecular weight 2000) with a maleic group on one end and NHS group onthe other end is used to conjugate to the aminal group of dioleyolphosphatidyl ethanolamine (DOPE). The resulting maleic-PEG-DOPE servesas a sulfhydryl-reactive lipid anchor with a peptide linker between thelipid portion and the SH-reactive group. Long-circulating liposomes areprepared by reverse phase evaportation method using a lipid mixturecomposed of DOPC:Cholesterol:PEG-DOPE:maleic-PEG-DOPE:Cy3-DOPE at aratio of 45:44:5:2:2 (molar ratio). The peptide is then conjugated tothe liposomes at pH 7.0 under inert gas for 24 hours at roomtemperature. After the conjugation, the excess of peptide is removedthough a gel filtration step using SEPHACRYL™-100 column with PBS aseluent. The percentage of conjugation of the peptide to the liposomes isestimated by the reduction of free peptide peak.

Experimental Design. HHLGGAKQAGDV (SEQ ID NO: 16) is conjugated toliposomes using SH-reactive group as described above. Liposomes arelabeled with gamma emitters and fluorochromes so that thepharmacokinetics and biodistribution can be measured.HHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-Liposomes are then coated with PEGas described above. Tumors are implanted and irradiated as describedabove. HHLGGAKQAGDV (SEQ ID NO: 16)-conjugated encapsulated drugs arethen injected by tail vein injection.

Biodistribution is studied by use of gamma emitters and gamma cameraimaging. Both large molecular weight proteins and small molecular weightcompounds (I.e. active agents) are radiolabeled. A therapeutic protein,tumor necrosis factor is labeled with ¹³¹I by use of IODO-GEN® reagentas described above. ¹³¹I-TNF is encapsulated in liposomes-HHLGGAKQAGDV(SEQ ID NO: 16) conjugates and PEG administered by tail vein asdescribed above.

Doxorubicin is used to study the biodistribution of a small MW compoundthat interacts with radiation. Doxorubicin is encapsulated influorescent liposomes (Avanti Polar Lipids) and PEG-HHLGGAKQAGDV (SEQ IDNO: 16) conjugates and administered by tail vein as described above.Methods of preparing fluorescent liposomes and conjugation ofHHLGGAKQAGDV (SEQ ID NO: 16) to liposomes are described above,Doxorubicin levels in serum and tumors in the Pharmacokinetic core labat Vanderbilt University using standard techniques. Fluorescencemicroscopy is used to measure liposomes in tumors using fluorescencequantification techniques described in Hallahan, 1997a.

Positive control groups. ¹³¹I-labeled HHLGGAKQAGDV-SGSGS-YYYYY (SEQ IDNO: 28) is administered to one group of irradiated tumor bearing miceand compared to biodistribution of encapsulated radiolabeled liposome.These mice are randomly assigned into groups during the same experimentas radiolabeled drugs. Radiolabeled drug binding in each group isquantified and compared to the ¹³¹I-labeled HHLGGAKQAGDV-SGSGS-YYYYY(SEQ ID NO: 28) positive control group.

Negative control groups: Firstly, control tumors are implanted in theleft hind limb of all mice and remain unirradiated. Secondly,SGSGSSGSGSGS-SGSGS (SEQ ID NO: 34) are conjugated to PEG and liposomesand administered to tumor bearing mice following irradiation to verifythat encapsulated drug binding to irradiated tumors is not a generalizedphenomenon. Eight tumor-bearing mice are randomly assigned into each ofthe experimental and control groups. Statistical considerations aredescribed above.

Example 14 Anti-P-Selectin scFv Binding to Microvasculature ofIrradiated Cancer

To determine whether anti-P-selectin scFv antibodies bind to irradiatedmicrovasculature, the binding of four antibodies (4A, 12F, 5H, and 10A)was studied using immunofluoresence microscopy. Human head and necksquamous cell carcinoma (HNSCC) cell lines were implanted into the hindlimb of nude mice and grown to 10 mm diameter as in Example 5 (see alsoHallahan et al., 1995b; Hallahan et al., 1998; Hallahan & Virudachalam,1999). Tumors were irradiated and dissected 5 hours later. Dissectedtumors were snap frozen and cryosectioned. Immunofluoresence microscopyof each of the scFv antibodies to human P-selectin demonstrated that theantigen in these tumor sections was expressed by host (mouse) cells,indicating that these epitopes are conserved across species. Each of thescFv antibodies bound to the microvasculature of irradiated HNSCC, butnot to untreated controls.

Example 15 Direct Application of Library to Irradiated Tumors andEndothelial Cells

To study the feasibility of selecting antibodies that bind irradiatedendothelial cells, primary culture human umbilical vein endothelialcells (HUVEC) were used. Negative selection of phage was first performedby removing all phage antibodies that bind within an intact umbilicalvein and to unirradiated endothelium from pooled donors. Unbound phagewere then incubated with HUVEC at 5 hours after irradiation with 2 Gy.Antibodies were prioritized by fluorometric microvolume assay technologywith an FMAT™ 8100 device (PE Biosystems, Foster City, Calif., UnitedStates of America) using irradiated HUVEC in microwells. Selected werescFv antibodies that bind with high affinity to irradiated HUVEC but donot bind to untreated HUVEC. Immunofluorescence microscopy of antibodiesdeveloped to irradiated HUVEC showed that several antibodies did notbind to untreated control cells but did bind to irradiated HUVEC. Thesephage-displayed antibodies were not displaced by anti-P-selectinantibodies indicating that they likely bound to distinctradiation-inducible epitopes on HUVEC. A determination of which of theseantibodies binds to human cancer microvasculature is presented inExample 17.

Phage antibodies that bind to irradiated HUVEC and fibroblasts using ahuman Fab antibody library are also selected. Enriched antibodies areprioritized and studied on biopsy specimens from irradiated HNSCCpatients. Antibodies that bind to human tumor blood vessels are isolatedand the radiation-inducible antigen(s) to which they bind arecharacterized. See Chang et al., 1991; Garrard et al., 1991; Hoogenboomet al., 1991; Kang et al., 1991; U.S. Pat. No. 5,837,500.

Example 16 In Vivo Testing of scFv Antibody Binding

Several scFv antibodies developed to P-selectin and to α_(2b)β₃ areprioritized by ELISA, BIACORE®, and fluorometric microvolume assaytechnology (the latter using a FMAT® 8100 device from PE Biosystems,Foster City, Calif., United States of America). These antibodies aretested to determine which bind to the greatest percentage of tumorspecimens from irradiated patients, while not binding to biopsies ofskin and mucosa. Biopsy specimens are sectioned on the day of antibodystaining, which is performed as described (Schueneman et al., 2003).Briefly, sections are first incubated with blocking buffer and washed.Fluorescence-labeled scFv and Fab antibodies are then incubated with thesections under conditions sufficient to allow binding of the antibodiesto targets. Antibody staining of tumor blood vessels is compared to thatof skin and mucosa biopsies from the same patients. Biopsies frompatients are stained for each of the prioritized antibodies by use offluorescence microscopy and image analysis software as has beendescribed (Geng et al., 2001; Hallahan et al., 2002). Vascular densityis also analyzed simultaneously.

HNSCC xenografts are implanted subcutaneously in the hind limb asdescribed in Hallahan et al., 2003. Antibodies and immunoconjugates withoptimal binding are radiolabeled and injected by tail vein afterirradiation of xenografts. The tumor bearing hind limb is irradiatedwith 0 Gy (Control), or daily fractionated radiation (2 Gy×7) asdescribed in Schueneman et al., 2003 and Hallahan et al., 2003.

Example 17 Mass Spectrometry Analysis of scFv Antibodies

To develop a high-throughput screening technique for phage libraryantibodies targeted to radiation-inducible neoantigens (for example,P-selectin or α_(2b)β₃ integrin) and measure tumor specificity of scFvantibodies developed from phage antibody libraries, a largephage-displayed scFv recombinant antibody library was developed. Thephage library was incubated with purified P-selectin protein, andhigh-affinity phage antibody clones were recovered by washing at pH 1.The antibody clones were assayed for antigen-binding activity by ELISA.The clones producing antibodies reactive with P-selectin were grown andinduced to express P-selectin-specific scFv antibodies on a large scale.

The phage antibody library was also screened for scFv that bound toexpired human platelets obtained from blood banks. Phage that werenonspecifically bound to inactivated platelets were first subtractedfrom the library. Platelets were activated to induce α_(2b)β₃ integrinin the active conformation. Bound phage were displaced by the additionof a monoclonal antibody specific for α_(1b). The displaced phage wererecovered and used to produce α_(1b) antibodies.

P-selectin and α_(2b) scFv antibodies were individually spotted inmatrix and evaluated by mass spectrometry for size to determine sets of6 that can be effectively discriminated by mass spectrometry based upondifferences in their molecular weights (approximately 400 mass unit sizedifference). Antibodies to P-selectin and to α_(2b) were administered insets of 6 by tail vein injection into mice bearing irradiated tumors.The tumors were dissected and antibody binding was measured by MALDI-TOFmass spectrometry.

Soluble rodent scFv antibodies to P-selectin and to α_(2b) weredeveloped, several of which were definitively measured in matrix byMALDI-TOF mass spectrometry. Of these, 9 soluble rodent scFv antibodiesto P-selectin and 9 soluble rodent scFv antibodies to α_(2b) weredifferentially detected in sets of 3 in mice tumors via MALDI-TOF massspectrometry. Spectrum analysis allowed quantification of the amount ofthe individual antibodies binding within the tumors.

Example 18 Binding of scFv to Human Cancer Microvasculature

Using the methods and procedures described hereinabove, scFv antibodiesthat are found to bind to HUVEC cells are tested for binding to humancancer microvasculature either in vivo or in vitro on biopsy samples.

Negative selection of the entire phage library (2×10⁹) is firstperformed on untreated vascular endothelium and platelets.Phage-displayed antibodies that bind to normal endothelium and plateletsare discarded, while phage that do not bind are used for high throughputscreening as follows.

HUVEC cells are grown to confluence in complete medium and human serumin 1536-well plates. Cells are irradiated with 3 Gy. Those scFv phageantibodies that bind to the isolated, irradiated endothelium areselected by use of an automated colony picker, followed by highthroughput screening using an FMAT® device (PE Biosystems, Inc., FosterCity, Calif., United States of America), which is used to quantifyfluorescence-labeled phage localized and concentrated on the irradiatedendothelial cell surface.

Example 19 Laser Capture Microdissection

Microvasculature is identified during laser capture microdissection(LCM). The use of an LCM system allows selected single cells or groupsof cells to be analyzed. LCM is used to dissect the vascular endotheliumand luminal proteins from a frozen section of an irradiated tumor. Thephage antibody library is added to these blood vessels and scFv phageantibodies that are recovered from the irradiated tumor vasculature areselected using an automated colony picker. Phage undergo several roundsof selection to reduce nonspecific binding. Identified antibodies arefurther selected using FMAT.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation—the presently disclosed subject matter beingdefined by the claims appended hereto.

What is claimed is:
 1. A method of detecting a tumor in a mammaliansubject, the method comprising: (a) exposing a suspected tumor toionizing radiation; (b) administering to the subject an immunoconjugatecomposition comprising a single chain fragment variable (scFv) antibodythat binds to a radiation-inducible neoantigen derived from tumorvascular endothelium selected from the group consisting of P-selectin,E-selectin, endoglin, α_(2b)β₃ integrin and α_(v)β₃ integrin, or a Fabfragment thereof, conjugated to a detectable label; and (c) detectingthe detectable label, whereby a tumor is detected.
 2. The method ofclaim 1, wherein the immunoconjugate is polyvalent.
 3. The method ofclaim 1, wherein the single chain fragment variable (scFv) antibody orFab antibody is humanized.
 4. The method of claim 1, wherein theimmunoconjugate is administered to the subject in a pharmaceuticallyacceptable carrier.
 5. The method of claim 1, further comprising adetectable label.
 6. The method of claim 1, wherein the detectable labelcomprises a label that can be detected using magnetic resonance imaging,scintigraphic imaging, ultrasound, or fluorescence.
 7. The method ofclaim 6, wherein the label that can be detected using scintigraphicimaging comprises a radionuclide label.
 8. The method of claim 1,wherein the radionuclide label comprises ¹³¹I or ⁹⁹mTc.